Complex formation between DSDNA and oligomer of cyclic heterocycles

ABSTRACT

Methods and compositions are provided for forming complexes between dsDNA and oligomers of heterocycles, aliphatic amino acids, particularly omega-amino acids, and a polar end group. By appropriate choice of target sequences and composition of the oligomers, complexes are obtained with low dissociation constants. The formation of complexes can be used for identification of specific dsDNA sequences, for inhibiting gene transcription, and as a therapeutic for inhibiting proliferation of undesired cells or expression of undesired genes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.08/837,524, filed Apr. 21, 1997, issued as U.S. Pat. No. 6,143,901,which is a continuation-in-part of application Ser. No. 08/607,078,filed Feb. 26, 1996, issued as U.S. Pat. No. 6,090,947 filed as a 371PCT application U.S. Pat. Ser. No. 97/03332, on Feb. 20, 1997, andprovisional application Ser. Nos. 60/023,309, filed on Jul. 31, 1996,now abandoned Ser. No. 60/024,374, filed on Aug. 1, 1996, now abandonedSer. No. 60/026,713 filed on Sep. 25, 1996, now abandoned and Ser. No.60/038,384, filed on Feb. 14, 1997.

The U.S. Government has certain rights in this invention pursuant toGrant Nos. GM 26453, 27681 and 47530 awarded by the National Instituteof Health.

BACKGROUND

With the explosion of techniques for the synthesis, analysis andmanipulation of nucleic acids, numerous new opportunities have arisen indiagnostics and therapeutics. In research there is substantial interestin being able to identify DNA sequences, which may be associated withspecific organisms, alleles, mutations, and the like, to understandparticular genetic processes, to identify diseases, for forensicmedicine, etc. Also, for many purposes, one may wish to modulate theactivity of a particular gene, so as to identify the function of aparticular gene, the effect of changes in its cellular concentration ofits gene product on the function of the cell, or other cellularcharacteristic. In therapeutics, one may wish to inhibit theproliferation of cells, such as bacterial, fungal and chlamydia cells,which may act as pathogens, of viruses, of mammalian cells, whereproliferation results in adverse effects on the host, or othersituation. In vivo, one may provide for reversible or irreversible knockout, so that information can be developed on the development of a fetus,or the effect on the organism of reduced levels of one or more geneticproducts.

In a number of seminal papers, Peter Dervan's group has shown thatoligomers of nitrogen heterocycles can be used to bind to dsDNA. It hasbeen shown that there is specificity in that G/C is complemented byN-methyl imidazole (Im)/ N-methyl pyrrole (Py), C/G is complemented byPy/Im, A/T and T/A are redundantly complemented by Py/Py. In effect,N-methyl imidazole tends to be associated with guanosine, while N-methylpyrrole is associated with cytosine, adenine, and thymidine. Byproviding for two chains of the heterocycles, as 1 or 2 molecules, a 2:1complex with dsDNA is formed, with the two chains of the oligomerantiparallel, where G/C pairs have Im/Py in juxtaposition, C/G pairshave Py/Im, and T/A pairs have Py/Py in juxtaposition. The heterocycleoligomiers are joined by amide carbamyl groups, where the NH mayparticipate in hydrogen bonding with nitrogen and oxygen unpairedelectrons of adenine and thymidine in the minor groove (FIG. 1),particularly of adenine. While the complexes were of substantialinterest, the binding affinities for the most part were less than about10⁶ M⁻¹. Furthermore, the discrimination between a target DNA sequence,and one involving a mismatch was frequently not better than abouttwo-fold. Therefore, for many purposes, the complexes had limitedutility.

Improvements in affinity were shown for a cyclic dimer, where the twooligomers were joined at their ends by γ-aminobutyric acid, where theaffinity was shown to be enhanced to about 10⁹M⁻¹. However, thedifference in affinity between the target sequence and were less thanthree-fold difference for three different single-base mismatchsequences. This low sequence-selectivity would severely limit theapplications for the compound in the presence of a large amount ofnaturally occurring dsDNA.

Also, for many applications, one wishes to be able to use the sequenceswith viable cells. There was no showing that these oligomers would becapable of being transported across a cellular membrane to the nucleusand, upon successful transport to the nucleus, they could bind to thechromosomal DNA, where the chromosomal DNA is present as nucleosomes.

RELEVANT LITERATURE

Wade et al., J.AM.CHEM.SOC., 1992, 114, 8783-8794; Mrkish et al.,PROC.NATL.ACAD.SCI. USA, 1992, 89, 7856-7590; Mrkish and Dervan, J.AM.CHEM. SOC., 1993, 115, 2572-2576; Wade et al., Biochemistry, 1993,32, 11385-11389; Mrkish and Dervan, J.AM.CHEM.SOC., 1993, 115,9892-9899; Dwyer et al., J.AM.CHEM.SOC., 1993, 115, 9900-9906; Mrkishand Dervan, J.AM.CHEM.SOC., 1994, 116, 3663-3664; Mrkish et al,J.AM.CHEM.SOC., 1994, 116, 7983-7988; Mrkish and Dervan J.AM.CHEM.SOC.,1995, 117, 3325-3332; Cho et al., PROC.NATL.ACAD.SCI. USA, 1995, 92,10389-10392; Geierstanger, Nature Structural Biology, 1996, 3, 321-324;Parks et al., J.AM.CHEM.SOC., 1996, 118, 6147-6152; Parks et al.,J.AM.CHEM.SOC., 1996, 118, 6153-6159; Baird and Dervan, J.AM.CHEM.SOC.,1996, 118, 6141-6146; Swalley et al., J.AM.CHEM.SOC., 1996, 118,8198-8206; Trauger et al., J.AM.CHEM.SOC., 1996, 118, 6160-6166;Szewczyk et al., J.AM.CHEM.SOC., 1996, 118, 6778-6779; Trauger et al.,Chemistry & Biology, 1996, 3, 369-377; Trauger et al., Nature, 1996,382, 559-561; Kelly et al., PROC.NATL.ACAD.SCI. USA, 1996, 93,6981-6985; Szewczyk et al., ANGEW.CHEM.INT.ED.ENGL., 1996, 35,1487-1489; Pilch et al., PROC.NATL.ACAD.SCI. USA, 1996, 93, 8306-8311;White et al., Biochemistry, 1996, 35, 12532-12537.

SUMMARY OF THE INVENTION

Methods and compositions are provided for selectively producing acomplex at a concentration of ≦1 nM, between dsDNA and an oligomer oforganic cyclic groups, wherein at least 60% of the cyclic groups areheterocyclics, and at least 60% of the heterocycles have at least onenitrogen annular member. The heterocycles form complementary pairs,where at least two of the nucleotide pairs are preferentially pairedwith a specific pair of heterocycles. There are at least threecomplementary pairs of organic cyclic groups in the complex, either as aresult of a hairpin turn in a single oligomer, or the complementationbetween organic cyclic groups of two oligomeric molecules. Usually, asmall aliphatic amino acid will be interspersed in or divide what wouldotherwise be a chain of six or more consecutive organic cyclic groups.To further enhance binding, a terminus may have at least one aliphaticamino acid of from two to six carbon atoms and/or an alkyl chain havinga polar group proximal to the linkage of the alkyl chain. By appropriateselection of the target sequence, the complementary pairs, unpairedorganic cyclic groups, the aliphatic amino acids, and thepolar-substituted alkyl chain, complexes may be formed with highaffinity, low dissociation constants, and significant disparities inaffinity between the target sequence and single-base mismatches.Modifications of the oligomers are used to provide for specificproperties and are permitted at sites which do not significantlyinterfere with the oligomers positioning in the minor groove. Thecompositions are found to be able to enter viable cells and inhibittranscription of genes comprising the target sequence, cleave atparticular sites, become covalently bonded at specific sites, directselected molecules to a target site, as well as perform other activitiesof interest.

The oligomers may be combined with dsDNA under complex formingconditions to form the complex. Formation of the complex can be used indiagnosis to detect a specific dsDNA sequence, where the oligomers maybe labeled with a detectable label, to reversibly or irreversibly “knockout” genes in vitro or in vivo, cytohistology, to inhibit proliferationof cells, both prokaryotic and eukaryotic, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B Model for Recognition of the DNA Minor-Groove. The DNAdouble helix consists of A,T and G,C base pairs like rungs on a twistedladder. Individual sequences may be distinguished by the pattern ofhydrogen bond donors and acceptors displayed on the edges of the basepairs. The A,T base pair presents two symmetrically placed hydrogen bondacceptors in the minor groove, the purine N3 and the pyrimidine O2 atomsrepresented as circles with dots. The G,C base pair presents these twoacceptors, but in addition presents a hydrogen bond donor, the 2-aminogroup of guanine represented as a circle containing an H. Because thehydrogen bond vector lies towards the guanine-containing strand, G.C andC.G base pairs may be distinguished in the minor groove. FIG. 1B PairingRules for DNA Recognition by Pyrrole-Imidazole for side-by-sidecomplexes Py/Impolyamides in the minor groove of DNA, the DNA bindingsequence specificity depends on the sequence of side-by-side amino acidpairings. A pairing of Im opposite Py targets a G.C base pair while apairing of Py opposite Im targets a C.G base pair. A Py/Py combinationis degenerate targeting both A.T and T.A base pairs. Specificity for G,Cbase pairs results from the formation of a putative hydrogen bondbetween the imidazole N3 and the exocyclic amino group of guanine.Putative hydrogen bonds are represented by dashed lines.

FIGS. 2A1, 2A2, 2B1, and 2B2 depict binding models for (a) 5′-AGTACT-3′(SEQ ID NO: 1) in complex with ImPyPyPy-γ-ImPyPyPy-β-Dp 1 (match) andImPyPyPy-γ-PyPyPy-β-Dp 2 (mismatch), and (b) 5′-AGTATT-3′ (SEQ ID NO: 2)in complex with ImPyPyPy-γ-PyPyPyPy-β-Dp 2 (match) andImPyPyPy-γ-ImPyPyPy-β-Dp 1 (mismatch). Circles with dots represents lonepairs on N3 of purines and O2 of pyrimidines, and circles containing Hrepresent the N2 hydrogen of guanine. Putative hydrogen bonds areillustrated by dashed lines. The dark and open circles representimidazole and pyrrole rings, respectively, the curved line representsγ-aminobutyric acid, and the diamond represents β-alanine. Singlehydrogen bond mismatches are highlighted.

FIG. 3A1(left): Model of nine zine finger protein TFIIIA with the 5S RNAgene internal control region (ICR). FIG. 3A1(right): Sequence of the ICRrecognized by zinc finger 4 in the minor groove. FIG. 3A2: Complex ofthe hairpin polyamide ImPyPyPy-γ-ImPyPyPy-β-Dp 1 with its target site,5′-AGTACT-3′ (SEQ ID NO: 1). Circles with dots represent lone pairs onN3 of purines and O2 of pyrimidines. Circles containing an H representthe N2 hydrogen of guanine. FIGS. 3B1 and 3B2: Structures of polyamidesImPyPyPy-γ-ImPyPyPy-β-Dp (1), ImPyPyPyPy-γ-PyPyPyPy-β-Dp (2), andImPyImPy-γ-PyPyPyPy-β-Dp (3). (Dp=dimethylaminopropylamide).

FIG. 3C: Binding models filled and open circles represent imidazole andpyrrole rings, respectively, the curved line represents γ-aminobutyricacid (γ), and the diamond represents β-alanine (β). Hydrogen bondmismatches are highlighted.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The subject invention provides novel oligomers for forming high affinitycomplexes with dsDNA. The oligomers comprise organic cyclic groupsjoined together by short linkers, which oligomers fit in the minorgroove of dsDNA and form complementary pairs with specific nucleotidebase pairs in the dsDNA target sequence. Associated with the organiccyclic compounds are aliphatic amino acids, particularly aliphatic aminoacids having a terminal amino group. In addition, a terminus willdesirably have a polar group, conveniently substituted on an alkylsubstituent. There will be a consecutive series of at least threecomplementary pairs of organic heterocycles, where by complementary isintended a preferential juxtaposition with a complementary pair ofnucleotides. By appropriate selection of complementary pairs, unpairedorganic cyclic compounds in juxtaposition to particular nucleotides ofbase pairs, aliphatic amino acids, and a polar group substituent, highaffinities and high specificities as compared to single-base mismatchescan be achieved. The subject compositions are shown to be capable ofbeing transported across cellular membranes to the nucleus, binding tochromosomal DNA, and fulfilling a variety of intracellular functions,including inhibiting transcription. The compositions may be modified tobe used in diagnostics, particularly by providing for detectable labels,or may be used in research or therapeutics, to inhibit transcription oftarget genes. The compositions may be otherwise modified to enhanceproperties for specific applications, such as transport across cellwalls, association with specific cell types, cleaving of nucleic acidsat specific sites, change chemical and physical characteristics, and thelike.

The oligomers of the subject invention will have at least six organicheterocyclic groups, more usually at least seven, and may have eight ormore, usually not more than about thirty, more usually not more thanabout twenty, frequently not more than about 18, organic cyclic groups,wherein at least 60%, preferably at least 80%, and more preferably atleast 100% are heterocycles. The heterocycles generally have from one tothree, more usually from one to two heteroatoms, where the heteroatomsare nitrogen, oxygen and sulphur, particularly nitrogen. The nitrogenatoms may be substituted, depending upon whether the nitrogen atom isdirected toward the floor or surface of the groove or away from thegroove. Greater latitude in the nature of the substitution is permittedwhen the nitrogen atom is directed away from the floor of the groove.The orientation of the oligomer is preferably N to C in association withthe 5′ to 3′ direction of the strand to which it is juxtaposed.

The heterocycles may be substituted at positions of the heterocyclewhich are directed away from the floor of the groove for any purpose.Thus, a hydrogen atom may be substituted with a substituent of interest,where the substituent will not result in steric interference with thewall of the minor groove or otherwise create repulsion. Whensubstituted, the substituents may be widely varied, being heteroatom,hydrocarbyl of from 1 to 30, more usually 1 to 20, carbon atoms,particularly 1 to 10, more particularly 1 to 6 carbon atoms, includingaliphatic, alicyclic, aromatic, and combinations thereof, including bothaliphatically saturated and unsaturated, having not more than 10% of thecarbon atoms participating in aliphatic unsaturation, heterosubstitutedhydrocarbyl (as defined previously), having from 1 to 10, usually 1 to8, more usually 1 to 6, heteroatoms, including aliphatic, alicyclic,aromatic and heterocyclic, and combinations thereof, where theheteroatoms are exemplified by halogen, nitrogen, oxygen, sulfur,phosphorous, metal atoms, boron, arsenic, selenium, rare earths, and thelike, wherein functional groups are exemplified by amino, includingmono- and disubstituted amino, oxy, including hydroxy and oxyether,thio, including mercapto and thioether, oxo, including oxo-carbonyl(aldehyde and ketone) and non-oxo-carbonyl (carboxy, including acylhalide, anhydride, ester, and amide), phosphorous, including phosphines,phosphites, phosphates, phosphoramidites, etc., boron, includingborates, borinic acids and borinates, nitro, cyano, azo, azoxy,hydrazino, etc.

The functional groups may be bonded to an annular member or to asubstituent bonded to an annular member, e.g. carboxyalkyl,methoxyethyl, methoxymethyl, aminoethyl, dialkylaminopropyl,polyoxyethylene, polyaminoethylene, etc. In many cases, for annularnitrogen substituents, conveniently, they will be substituted with analkyl group of from 1 to 3 carbon atoms, particularly methyl, and atleast one adjacent annular carbon atom unsubstituted. For the most part,individual substituents will be under 600 Dal, usually under about 300Dal, and preferably under about 150 Dal and the total for substituentsbonded to annular members will be under about 5 kDal, usually underabout 2 kDal, more usually under about 1 kdal, there generally beingfrom about 0 to 5, more usually from about 0 to 3 substituents, forother than the alkyl of from 1 to 3 carbon atoms bonded to annularnitrogen. Generally, the total carbon atoms for the substituents willnot be greater than about 100, usually not greater than about 60, moreusually not greater than about 30, with not more than 30 heteroatoms,usually not more than 20 heteroatoms, more usually not more than about10 heteroatoms.

The heterocycles will normally be linked at the 2 position and the 4 or5 position, particularly the 2 and 4 position for 5 annular memberrings.

The heterocycles are five to six annular members, particularly fiveannular members, having from one to three, usually one to twoheteroatoms, where two heteroatoms are usually spaced apart by at leastone intervening carbon atom. The organic cyclic groups are completelyunsaturated and will be referred to as aromatic as that term isunderstood for organic cyclic compounds of from five to six annularmembers.

Illustrative annular members include pyrrole, imidazole, triazole,furan, thiophene, oxazole, thiazole, pyrazole, cyclopentadiene,pyridine, pyrimidine, triazine, and the like, where as indicated above,NH groups in the rings when substituted are preferably alkylated with analkyl group of from one to three carbon atoms, particularly methyl. Thepreferred organic cyclic compounds are five membered rings having fromone to two nitrogen atoms, where one of the nitrogen atoms ismethylated.

The linking groups between the organic cyclic groups will generally havea length of two atoms, wherein at least some of the linking groups willhave NH, where the NH may hydrogen bond with an unshared pair ofelectrons of the nucleotides. The linking chains may be methyleneamino,carbamyl (—CONH—), ethylene, thiocarbamyl, imidinyl, and the like,particularly carbamyl and its heteroanalogs, e.g. thio and imino.

In addition to the organic cyclic compounds, aliphatic amino acids areemployed, particularly ω-amino aliphatic amino acids, either to providefor hairpin turns to provide complementation between two sequences ofheterocycles, to form a cyclic compound where the oligomers are joinedat both ends, or to provide for a shift in spacing of the organic cycliccompounds in relation to the target dsDNA. For the most part, thealiphatic amino acids will have a chain as a core structure of two tosix carbon atoms, usually of two to four carbon atoms, desirably havingterminal amino groups, particularly glycine, β-alanine, andγ-aminobutyric acid, being unsubstituted or substituted on carbon andnitrogen, particularly carbon, although for the most part the aliphaticamino acids will be unsubstituted. The substituents have been describedpreviously. Where an aliphatic amino acid is C-terminal, the carboxylgroup will usually be functionalized as an ester or amide, where thealcohol or amino acid may be selected to provide for specific propertiesor be used to reduce the charge of the carboxyl group. For the latter,the alcohol and amino groups will generally be from 0 to 6 carbon atoms,usually from 0 to 3 carbon atoms.

As indicated above, these amino acids will play specific roles. Thelonger chain aliphatic amino acid will serve to provide for turns in themolecule and to close the molecule to form a ring. The shorter chainaliphatic amino acids will be employed, both to provide a shift forspacing in relation to the target dsDNA, and to provide enhanced bindingby being present proximal to the terminal organic cyclic group. Thealiphatic amino acid may be present at one or both ends of the oligomer.Of particular interest are glycine and alanine, for space-shifting,β-alanine is preferred. Usually, a consecutive sequence of 6heterocycles will be avoided. Generally, there will be an amino acid,particularly β-alanine, introduced in an otherwise consecutive series ofsix oligomer units, generally bordered by at least one, preferably atleast two organic cyclic groups, particularly heterocycles. Thefollowing table indicates the effect of extension of the oligomerheterocycles without introducing an amino acid in the chain.

TABLE 1* binding site specificity polyamide sub-unit size, bp matchmis-match ˜ Im-(Py)₂-Dp 3 5 1.3 × 10⁵ (0.3)  <2 × 10^(4′) >6.5^(&)Im-(Py)₃-Dp 4 6 8.5 × 10⁶ (1.3) 1.6 × 10⁶ (0.2) 5.3 (0.5) Im-(Py)₄-Dp 57 4.5 × 10⁷ (1.1) 7.9 × 10⁶ (1.8) 5.7 (0.8) Im-(Py)₅-Dp 6 8 5.3 × 10⁷(0.5)  <2 × 10^(7′) >2.7^(&) Im-(Py)₆-Dp 7 9 4.7 × 10⁷ (0.4) 1.7 × 10⁷(0.7) 2.8 (0.7) Im-(Py)₇-Dp 8 10  <2 × 10⁷  <2 × 10^(6′) ˜1 *Valuesreported are the mean values from at least three footprint titrationexperiments. Numbers in parentheses indicate the standard deviation foreach data set. The assays were performed at 22° C., pH 7.0, in thepresence of 10 mM TrisHCl, 10 mM KCl, 10 mM MgCl₂ and 5 mM CaCl₂.˜Defined as the ratio of the match site affinity to the affinity of thesingle base pair mismatch site. Numbers in parentheses indicate theuncertainty calculated using the standard deviations of the measuredbinding affinities. ^(&)Represents a lower limit on the specificity.^(′)Represents an upper limit for the binding affinity.

The aliphatic chains of the aliphatic amino acids may serve as sites ofsubstitution, the aliphatic amino acid providing a core structure, thereusually being not more than 2, more usually not more than 1,substituent. The same types of substituents that have been described forthe heterocycles may also be employed here. Conveniently, thesubstituted aliphatic amino acid may be used in the synthesis of theoligomer, rather than modifying the amino acid after the oligomer isformed. Alternatively, a functional group may be present on the chain ofthe substituent, if necessary being appropriately protected during thecourse of the synthesis, which functional group may then be used for thesubsequent modification. Desirably, such functional group could beselectively used, for synthesis of different oligomers, so as to providefor substitution at that site to produce products having uniqueproperties associated with a particular application. With thesubstituent substituted at a site which does not significantly interferewith the binding in the groove, e.g. employing a single stereoisomer,properties can be imparted to the subject compounds, such as watersolubility, lipophilicity, non-covalent binding to a receptor,radioactivity, fluorescence, etc.

One or both termini, preferably one of the termini, will have a polargroup substituted on an alkyl group, where the polar group willgenerally be from 2 to 6, more usually 2 to 4, carbon atoms from thelinkage to the remaining molecule. The polar group may be charged oruncharged, where the charge may be a result of protonation under theconditions of use. Particularly, groups capable of hydrogen bonding arepreferred, such as amino, particularly tertiary-amino, hydroxyl,mercapto, and the like. Of particular interest is amino, moreparticularly alkylated amino, where the alkyl groups are of from 1 to 6,usually 1 to 3, more usually 1, carbon atom, and at a pH less than abouteight, the amino group is positively charged, and can hydrogen bond withthe dsDNA. Desirably, two positively charged polar groups will not beemployed on the oligomers, where the positively charged polar groupswill be in juxtaposition when complexed with the dsDNA.

For many purposes one may wish to have an isotopic oligomer, where onecan analyze for its presence, using scintillation counters forradioactive elements, nmr for atoms having a magnetic moment, and thelike. For a radioactive oligomer, a radioactive label may be employed,such as tritium, ¹⁴C, ¹²⁵I, or the like. The radiolabel may be asubstituent on an annular member of a heterocycle or an annular memberof a heterocycle, either carbon or a heteroatom, or a substituent at theC- or N-terminus of the oligomer, depending upon convenience. By using aradiolabel as part of the oligomer, one avoids any significant change inthe spatial conformation of the oligomer. The radiolabel may servenumerous purposes in diagnostics, cytohistology, radiotherapy, and thelike.

Besides the other sites present on the oligomer, either terminus of theoligomer may be used for special purposes depending upon the use towhich the oligomer is put. For example, in diagnostics, one may wish tohave a detectable label other than a radiolabel, where the resultingcompound may find use for other purposes, as well. The oligomer may belinked to labels, such as fluorescers, e.g. dansyl, fluorescein, Texasred, isosulfan blue, ethyl red, malachite green, etc., chemiluminescers,particles, e.g. magnetic particles, colloidal particles, e.g. goldparticles, light sensitive bond forming compounds, e.g. psoralens,anthranilic acid, pyrene, anthracene, and acridine, chelating compounds,such as EDTA, NTA, tartaric acid, ascorbic acid, polyhistidines of from2 to 8 histidines, alkylene polyamines, etc., chelating antibiotics,such as bleomycin, where the chelating compounds may chelate a metalatom, such as iron, cobalt, nickel, technetium, etc., where the metalatom may serve to cleave DNA in the presence of a source of peroxide,intercalating dyes, such as ethidium bromide, thiazole orange, thiazoleblue, TOTO, 4′,6-diamidino-2-phenylindole (DAPI), etc., enzymes, such asβ-galactosidase, NADH or NADHP dehydrogenase, malate dehydrogenase,lysozyme, peroxidase, luciferase, etc., alkylating agents such ashaloacetamides, N-ethyl nitrosourea, nitrogen and sulfur mustards,sulfonate esters, etc., and other compounds, such as arylboronic acids,tocopherols, lipoic acid, captothesin, etc. colloidal particles, e.g.gold particles, fluorescent particles, peroxides, DNA cleaving agents,oligonucleotides, oligopeptides, nmr agents, stable free radicals, metalatoms, etc. The oligomer may be combined with other labels, such ashaptens for which a convenient receptor exists, e.g. biotin, which maybe complexed with avidin or streptavidin and digoxin, which may becomplexed with antidigoxin, etc. where the receptor may be conjugatedwith a wide variety of labels, such as those described above. Theoligomers may be joined to sulfonated or phosphonated aromatic groups,e.g. naphthalene, to enhance inhibition of transcription, particularlyof viruses (Clanton et al., Antiviral Res. (1995) 27:335-354). In someinstances, one may bond multiple copies of the subject oligomers topolymers, where the subject oligomers are pendant from the polymer.Polymers, particularly water soluble polymers, which may find use arecellulose, poly(vinyl alcohol), poly(vinyl acetate-vinyl alcohol),polyacrylates, and the like. The number of oligomers may be from 1 toabout 1:5 monomer units of the polymer.

One may wish to enhance the lipophilicity of the molecule, providing forvarious lipophilic groups, such as cholesterol, fatty acids, fattyalcohols, sphingomyelins, cerebrosides, other glycerides, and the like,where the fatty group will generally be of from about eight to thirtycarbon atoms. Alternatively, one may wish to provide for saccharides,which bind to lectins, adhesion molecules, bacteria, or the like, wherethe saccharides serve to direct the subject oligomers to a specificcellular target. Alternatively, in some instances, one may wish to haveone or more nucleotides, generally from about one to thirty, moreusually from about three to twenty, particularly from about three totwelve. The nucleotides will normally be associated with the proximal orbordering nucleic acid sequence of the target sequence, whereby theattached nucleic acid sequence will complex with the nucleotides in themajor groove.

The different molecules may be joined to the termini in a variety ofways, depending upon the available functionality(ies) present at thetermini, such as extending the polar substituted alkyl group, e.g.having a chain of more than 6 carbon atoms, providing for a substituentat a terminus which can be reacted with the moiety to be added, wheresuch substituents will conventionally be amino, hydroxyl, mercapto,carboxyl, phosphate, etc., so as to form amides, both organic andinorganic, substituted amines (reductive amination), ethers, thioethers,disulfides, esters, both organic and inorganic, pyrophosphates, and thelike. The molecules may be introduced as part of the synthetic scheme,displacing the oligomer from the solid support on which the oligomer issynthesized. Because the compounds of the subject invention may be usedin such a variety of ways, no simple description is appropriate to thevariety of moieties to which the subject oligomers may be bound, nor thespecific molecular weights of the resulting products.

The subject oligomers may be synthesized on supports, e.g. chips, whereby using automated synthetic techniques, different oligomers may besynthesized at individual sites. In this way, an array of differentoligomers may be synthesized, which can then be used to identify thepresence of a plurality of different sequences in a sample. By knowingthe composition of the oligomer at each site, one can identify bindingof specific sequences at that site by various techniques, such aslabeled antiDNA antibodies, linkers having complementary restrictionoverhangs, where the sample DNA has been digested with a restrictionenzyme, and the like. The techniques for preparing the subject arraysare analogous to the techniques used for preparing oligopeptide arrays,as described in Cho et al., Science, 1993, 261, 1303-1305.

The complex will usually comprise one or two oligomers or combinationsof one or two oligomers, where individual or pairs of oligomersspecifically interact with a dsDNA sequence of at least 6, usually atleast 7 and preferably 8 or more bp, frequently not more than 40 bp,more usually not more than about 30 bp, preferably not more than 20 bp.

Since a major portion of the work has been performed with N-methylpyrrole and N-methyl imidazole, using carbamyl groups as the linkingchains, with the aliphatic amino acids glycine, 13-alanine andγ-aminobutyric acid, as well as dimethylaminopropyl as the polarsubstituted alkyl group, these compounds will now be illustrated asexemplary of the class of compounds which may be employed in the subjectinvention. It is understood that one or a few of thenitrogen-heterocycles may be substituted with a different organic cyclicgroup, as well one or the other of the aliphatic amino acids may besubstituted with a different amino acid, etc. Furthermore, the coreoligomer may be further substituted for specific applications asdescribed above. In effect, there is a core molecule or core moleculeswhich define at least complementary pairs of heterocycles, and includeat least one of an aliphatic amino acid and a polar group substitutedalkyl. This core molecule which is the centerpiece of the invention canserve as the nexus for numerous substitutions which do not interferewith the basic function of the core molecule, although where the bindingaffinity is greater than is necessary for the function, some degradationof the binding affinity is permitted. Therefore, in defining thecompounds of this invention, it should be understood that manyvariations are permitted, where the basic core or unit structure isretained, while the core or unit structure is modified with one or moresubstituents to impart desired properties to the molecule for itsintended function.

Of particular interest among the subject compounds are compounds whichhave at least one organic cyclic group, particularly N-methyl imidazole,which has specificity for one nucleotide, which is present as acomplementary pair. Usually, the subject compounds will have at leastone of these complementary pairs, frequently at least two of thesecomplementary pairs, and generally fewer than 75% of the complementarypairs will have the organic cyclic group having specificity for a singlenucleotide. In the case of the N-methyl imidazole, there will usually beat least one Im/Py pair, desirably not having more than three,frequently having not more than two, of such pairs consecutively, sothat there will frequently be not more than three Im's in a row. Therewill normally be at least one aliphatic amino acid, frequently twoaliphatic amino acids, and frequently not more than eight aliphaticamino acids, usually not more than six aliphatic amino acids, moreusually not more than about four aliphatic amino acids. Preferably,there will be an amino acid proximal to at least one terminus of theoligomer. The Im/Py pair provide for greater specificity, and whenappropriately placed contribute in at least a similar manner to thePy/Py pair to the binding affinity for dsDNA. Therefore, by appropriateselection of the target sequence, one may optimize for binding affinityand specificity.

It is found that with β-alanine, β-alanine associates with T-A pairs andwill usually form a complementary pair with itself. Thus, β-alanine maybe used in juxtaposition to T or A and as a complementary pair withitself with a T-A pair.

The binding affinity K_(a) as determined in the Experimental sectionwill be greater than 5×10⁸ M⁻¹, usually greater than 10⁹ M⁻¹, preferablygreater than about 10¹⁰ M⁻¹, so as to be able to bind to the targetsequence at subnanomolar concentrations in the environment in which theyare used. The difference in affinity with a single mismatch will be atleast 3 fold, usually at least 5 fold, preferably at least 10 fold andfrequently greater than 20 fold, and may be 100 fold or more.

Where the oligomers of the subject invention are used with cells,particularly viable cells, the oligomers will generally have a molecularweight of less than about 5 kD, preferably less than about 3.5 kD, andwill generally have a molecular weight of at least about 0.6 kD, moreusually at least about 0.8 kD.

The compositions of the subject invention for complexing with dsDNA willhave from one to two oligomers, or combinations thereof, depending uponwhether there is a hairpin turn in the oligomer, where only one oligomeris necessary, or there is no hairpin turn, so that for complementarity,one needs two oligomers. More oligomers may be used, where one wishes totarget more than one dsDNA sequence, for example, contiguous or proximalsequences, to enhance the overall specificity, or for distal sequences,where the sequences may be associated with the same functional unit,e.g. a gene, or different functional units, e.g. homeodomains. Thecomposition, whether a single oligomer or a combination of oligomerswill provide at least three complementary pairs in the single oligomeror pair of oligomers.

In many cases, in order to achieve the desired association constants,one will increase the number of complementary pairs and/or have regionsof unpaired organic cyclic groups. Usually, one will have at least oneor both of a fourth complementary pair or at least two unpaired organiccyclic groups, so as to have a chain of four organic cyclic groupsinvolved in pair formation and/or at least two organic cyclic groupsuninvolved with pair formation. It is found that one does not increasethe binding affinity to the same extent with each addition of an organiccyclic unit, as one extends the length of the oligomer and, in fact, asdescribed previously, one may begin to reverse the binding affinity bythe continuous extension. Therefore, by appropriate choice, as indicatedabove, one can limit the composition and size of individual oligomers tooptimize the binding affinity, as well as the other properties which areassociated with the oligomeric composition.

Because of the extensive utilization of N-methyl pyrrole and N-methylimidazole, the following compounds which employ these N-heterocycles areexemplary of the class of compounds of the subject invention. Thesecompounds may be prepared in accordance with the procedures describedherein. When used, Py will refer to N-methyl pyrrole and Im will referto N-methyl imidazole.

ImPyPyPy-γ-PyPyPyPy, PyPyImPy-γ-PyPyPyPy, ImPyPyPy-γ-ImPyPyPy,PyImPyPy-γ-PyImPyPy, ImPyImPy-γ-PyPyPyPy, ImImPyPy-γ-PyPyPyPy,ImImImPy-γ-PyPyPyPy, ImImPyPy-γ-ImPyPyPy, ImPyPyPy-γ-ImImPyPy,ImImPyPy-γ-ImImPyPy, ImPyImPy-γ-ImPyImPy, ImImImPy-γ-ImPyPyPyPy,ImImImIm-γ-PyPyPyPy, Im-β-PyPy-γ-Im-β-PyPy, Im-β-ImIm-γ-Py-β-PyPy,Im-β-ImPy-γ-Im-β-ImPy, ImPyPyPyPy-γ-ImPyPyPyPy, ImImPyPyPy-γ-ImPyPyPyPy,ImPyImPyPy-γ-ImPyPyPyPy, ImImPyImIm-γ-PyPyPyPyPy,ImPyPyImPy-γ-ImPyPyImPy, ImPy-β-PyPy-γ-ImPy-β-PyPy,ImIm-β-ImIm-γ-PyPy-β-PyPy, ImPy-β-ImPy-γ-ImPy-β-ImPyImPy-β-PyPyPy-γ-ImPyPy-β-PyPy, ImIm-β-PyPyPy-γ-PyPyPy-β-PyPy,ImPy-β-ImPyPy-γ-ImPyPy-β-PyPy, ImIm-β-PyPyPy-γ-ImImPy-β-PyPy,ImPy-β-PyPyPy-γ-PyPyPy-β-ImPy, ImPyPyPyPyPy-γ-ImPyPyPyPyPy,ImPyPy-β-PyPy-γ-ImPyPy-β-PyPy, ImPyPyPy-β-Py-γ-Im-β-PyPyPyPy,ImImPyPyPyPy-γ-ImImPyPyPyPy, Im-β-PyPyPyPy-γ-Im-β-PyPyPyPy,ImPyPyPy-β-Py-γ-ImPyPyPy-β-Py, ImPyImPyPyPy-γ-ImPyPyPyPyPy,ImPyPy-β-PyPy-γ-ImPy-β-PyPyPy, ImPyPyPyPy-β-γ-ImPyPyPyPy-β,ImPy-β-ImPyPy-γ-ImPy-β-ImPyPy, Im-β-PyPyPyPy-γ-ImPyPyPy-β-Py,Im-β-ImPyPyPy-γ-ImPyPyPy-β-Py, ImPyPy-β-PyPyPy, ImImPy-β-PyPyPy,ImImIm-β-PyPyPy, ImPyPyPyPy-β-PyPyPy, ImPyPyPy-β-PyPyPy,ImPyPy-β-PyPyPyPyPy, ImPyPyPy-β-PyPyPyPy, ImImPyPy-β-PyPyPyPy,ImImImPy-β-PyPyPyPy, ImPyPyPy-β-ImPyPyPy, ImImPyPy-β-ImPyPyPy,ImImPyPyPy-β-PyPyPyPy, ImImImPyPy-β-PyPyPyPyPy,ImIm-β-PyPy-β-PyPy-β-PyPy, ImImPy-β-PyPyPy-β-PyPyPy,ImImPyPy-β-Py-β-PyPyPyPy, ImPyPy-γ-ImPyPy-β-PyPyPy,ImPyPy-γ-PyPyPy-β-PyPyPy, PyImPy-γ-ImPyPy-β-PyPyPy,PyImPy-γ-ImPyPy-β-PyPyPy-β-PyPyPy, ImImPy-γ-ImPyPy-β-PyPyPy,ImPyPy-γ-ImPyPy-G-PyPyPy, ImPyPyPy-γ-ImImIm-β-PyPyPyPy,ImImPyPy-γ-ImImPyPy-β-PyPyPyPy, and ImImPyPy-γ-PyPyPyPy-β-PyPyPyPy,PyPyImIm, ImPy-β-ImPy-β-ImPy, ImPy-β-ImPy-β-ImPy-β-PyPy,ImPy-β-ImPy-β-ImPy-γ-ImPy-β-ImPy-β-ImPy, ImPy-β-PyPy-β-PyPy,ImPy-β-PyPy-β-PyPy-γ-ImPy-β-PyPy-β-PyPy, Im-β-ImImImIm-γ-PyPyPyPy-β-Py,Im-β-ImImImIm-β-Im-γ-Py-β-PyPyPyPy-β-Py, ImIm-β-Im-γ-Py-β-PyPyPyPy-β-Py,Im-β-ImPyPy-γ-ImImPy-β-Py, ImImPy-β-Py-γ-Im-β-ImPyPy,ImIm-β-Im-γ-PyImPyPy.

FIG. 1 illustrates the relationship between the azoles and thenucleotides of the minor groove.

Where two oligomers are used, the oligomers may be completelyoverlapped, or only partially overlapped, i.e. slipped or haveoverhangs. As indicated previously, there will be at least 3complementary azole (N-methyl pyrrole and imidazole)pairs. In theoverlapped configuration, all of the azoles are in complementary pairs,as well as any spacing amino acid. In the slipped configuration, therewill be at least one azole ring which is unpaired in at least one of theoligomers, usually there will be at least two azole rings, more usually,in both of the oligomers. Usually, the number of unpaired azole ringswill be in the range of 2 to 30, more usually 2 to 20, frequently 2 to12. Generally, unpaired azoles will involve chains of 2 or more azolerings, more usually 3 or more azole rings, including, as appropriate,aliphatic amino acids in the chain.

Various permutations and combinations of oligomers may be used. One mayhave a single oligomer having at least three complementary pairs and anextension or overhang of unpaired azoles, which may be complemented inwhole or part by a second oligomer, which forms complementary pairs withthe unpaired members of the first oligomer. Alternatively, one may havetwo “candy cane” oligomers, having complementary pairs, with the membersof the complementary pairs separated by a γ-aminobutyric acid, and anoverhang of unpaired members. However, these otherwise unpaired membersof one oligomer can be positioned to form complementary pairs with theextension or overhang of the unpaired members of the other oligomer. Onemay have an extended linear oligomer, where two or more oligomerscomplement the azoles of the extended linear oligomer. If one wished,one could have alternating regions of unpaired and paired azoles byusing a plurality of oligomers which complement to various degrees. Ineach case, the selection would be related to the desired affinity, thenature of the target, the purpose for the formation of the complex, andthe like.

The subject compositions may be brought together with the dsDNA under avariety of conditions. The conditions may be in vitro, in cell cultures,ex vivo or in vivo. For detecting the presence of a target sequence, thedsDNA may be extracellular or intracellular. When extracellular, thedsDNA may be in solution, in a gel, on a slide, or the like. The dsDNAmay be present as part of a whole chromosome or fragment thereof of oneor more centiMorgans. The dsDNA may be part of an episomal element. ThedsDNA may be present as smaller fragments ranging from about 20, usuallyat least about 50, to a million base pairs, or more. The dsDNA may beintracellular, chromosomal, mitochondrial, plastid, kinetoplastid, orthe like, part of a lysate, a chromosomal spread, fractionated in gelelecrophoresis, a plasmid, or the like, being an intact or fragmentedmoiety. The formation of complexes between dsDNA and the subjectcompounds may be for diagnostic, therapeutic, purification, or researchpurposes, and the like. Because of the specificity of the subjectcompounds, the subject compounds can be used to detect specific dsDNAsequences in a sample without melting of the dsDNA. The diagnosticpurpose for the complex formation may be detection of alleles,identification of mutations, identification of a particular host, e.g.bacterial strain or virus, identification of the presence of aparticular DNA rearrangement, identification of the presence of aparticular gene, e.g. multiple resistance gene, forensic medicine, orthe like. With pathogens, the pathogens may be viruses, bacteria, fungi,protista, chlamydia, or the like. With higher hosts, the hosts may bevertebrates or invertebrates, including insects, fish, birds, mammals,and the like or members of the plant kingdom.

When involved in vitro or ex vivo, the dsDNA may be combined with thesubject compositions in appropriately buffered medium, generally at aconcentration in the range of about 0.1 nM to 1 mM. Various buffers maybe employed, such as TRIS, HEPES, phosphate, carbonate, or the like, theparticular buffer not being critical to this invention. Generally,conventional concentrations of buffer will be employed, usually in therange of about 10-200 mM. Other additives which may be present inconventional amounts include sodium chloride, generally from about 1-250mM, dithiothreitol, and the like, the particular nature of quanitity ofsalt not being critical to this invention. The pH will generally be inthe range of about 6.5 to 9, the particular pH not being critical tothis invention. The temperature will generally be in a range of 4° C. to45° C., the particular temperature not being critical to this invention.The target dsDNA may be present in from about 0.001 to 100 times themoles of oligomer.

The subject compounds when used in diagnosis may have a variety oflabels as indicated previously and may use many of the protocols thathave been used for detection of haptens and receptors (immunoassays) orwith hybridization (DNA complementation). Since the subject compoundsare not nucleic acids, they can be employed more flexibly than whenusing DNA complementation. The assays are carried out as described belowand then depending on the nature of the label and protocol, thedetermination of the presence and amount of the sequence may then bemade. The protocols may be performed in solution or in association witha solid phase. The solid phase may be a vessel wall, a particle, fiber,film, sheet, or the like, where the solid phase may be comprised of awide variety of materials, including gels, paper, glass, plastic,metals, ceramics, etc. Either the sample or the subject compounds may beaffixed to the solid phase in accordance with known techniques. Byappropriate functionalization of the subject compounds and the solidphase, the subject compounds may be covalently bound to the solid phase.The sample may be covalently or non-covalently bound to the solid phase,in accordance with the nature of the solid phase. The solid phase allowsfor a separation step, which allows for detection of the signal from thelabel in the absence of unbound label.

Exemplary protocols include combining a cellular lysate, with the DNAbound to the surface of a solid phase, with an enzyme labeled oligomer,incubating for sufficient time under complex forming conditions for theoligomer to bind to any target sequence present on the solid phase,separating the liquid medium and washing, and then detecting thepresence of the enzyme on the solid phase by use of a detectablesubstrate.

A number of protocols are based on having a label which does not give adetectable signal directly, but relies on non-covalent binding with areceptor, which is bound to a surface or labeled with a directlydetectable label. In one assay one could have a hapten, e.g. digoxin,bonded to the oligomer. The sample DNA would be bound to a surface, soas to remain bound to the surface during the assay process. The oligomerwould be added and bind to any target sequence present. After washing toremove oligomer, enzyme or fluorescer labeled antidigoxin monclonalantibody is added, the surface washed and the label detected.Alternatively, one may have a fluorescer bound to one end of theoligomer and biotin or other appropriate hapten bound to the other endof the oligomer or to the complementary oligomer. The oligomers arecombined with the DNA in the liquid phase and incubated. Aftercompletion of the incubation, the sample is combined with the receptorfor the biotin or hapten, e.g. avidin or antibody, bound to a solidsurface. After a second incubation, the surface is washed and the levelof fluorescence determined.

If one wishes to avoid a separation step, one may use channeling orfluorescence quenching. By having two labels which interact, forexample, two enzymes, where the product of one enzyme is the substrateof the other enzyme, or two fluorescers, where there can be energytransfer between the two fluorescers, one can determine when complexformation occurs, since the two labels will be brought intojuxtaposition by forming the 2:1 complex in the minor groove. With thetwo enzymes, one detects the product of the second enzyme and with thetwo fluorescers, one can determine fluorescence at the wavelength of theStokes shift or reduction in fluorescence of the fluorescer absorbinglight at the lower wavelength. Another protocol would provide forbinding the subject compositions to a solid phase and combining thebound oligomers with DNA in solution. After the necessary incubationsand washings, one could add labeled antiDNA to the solid phase anddetermine the amount of label bound to the solid phase.

To determine a number of different sequences simultaneously or just asingle sequence, one may provide an array of the subject compositionsbound to a surface. In this way specific sites in the array will beassociated with specific DNA sequences. One adds the DNA containingsample to the array and incubates. DNA which contains the complementarysequence to the oligomer at a particular site will bind to the oligomersat that site. After washing, one then detects the presence of DNA atparticular sites, e.g. with an antiDNA antibody, indicating the presenceof the target sequence. By cleaving the DNA with a restriction enzyme inthe presence of a large amount of labeled linker, followed byinactivation of the enzyme, one may then ligate the linker to thetermini of the DNA fragments and proceed as described above. Thepresence of the label at a particular site in the array will indicatethe presence of the target sequence for that site.

The number of protocols that may be used is legion. Illustrativeprotocols may be found for DNA assays in WO95/20591; EPA 393,743;WO86/05519; and EPA 278,220, while protocols and labels which may beadapted from immunoassays for use with the subject compositions forassays for DNA may be found in WO96/20218; WO95/06115; WO94/04538;WO94/01776; WO92/14490; EPA 537830; WO91/09141; WO91/06857; andWO91/05257.

During diagnostics, such as involved with cells, one may need to removethe non-specifically bound oligomers. This can be achieved by combiningthe cells with a substantial excess of the target sequence, convenientlyattached to particles. By allowing for the non-specifically boundoligomers to move to the extracellular medium, the oligomers will becomebound to the particles, which may then be readily removed. If desired,one may take samples of cells over time and plot the rate of change ofloss of the label with time. Once the amount of label becomesstabilized, one can relate this value to the presence of the targetsequence. Other techniques may also be used to reduce false positiveresults.

The subject compositions may also be used to titrate repeats, wherethere is a substantial change, increase or decrease, in the number ofrepeats associated with a particular indication. The number of repeatsshould be at least an increase of 50%, preferably at least two-fold,more preferably at least three-fold. By determining the number ofoligomers which become bound to the dsDNA, one can determine theamplification or loss of a particular repeat sequence.

The subject compositions may be used for isolation and/or purificationof target DNA comprising the target sequence. By using the subjectoligomers, where the oligomers are bound to a solid phase, thoseportions of a DNA sample which have the target sequence will be bound tothe subject oligomers and be separated from the remaining DNA. One canprepare columns of particles to which the oligomers are attached andpass the sample through the column. After washing the column, one canrelease the DNA which is specifically bound to the column using solventsor high salt solutions. Alternatively, one can mix particles to whichthe oligomers are bound with the sample and then separate the particles,for example, with magnetic particles, using a magnetic field, withnon-magnetic particles, using centrifugation. In this way, one canrapidly isolate a target DNA sequence of interest, for example, a genecomprising an expressed sequence tag (EST), a transcription regulatorysequence to which a transcription factor binds, a gene for which afragment is known, and the like. As partial sequences are defined by avariety of techniques, the subject oligomers allow for isolation ofrestriction fragments, which can be separated on a gel and thensequenced. In this way the gene may be rapidly isolated and its sequencedetermined. As will be discussed below, the subject oligomers may thenbe used to define the function of the gene.

The subject oligomers may be used in a variety of ways in research.Since the subject oligomers can be used to inhibit transcription, theeffect of inhibiting transcription on cells, cell assemblies and wholeorganisms may be investigated. For example, the subject compositions maybe used in conjunction with egg cells, fertilized egg cells orblastocysts, to inhibit transcription and expression of particular genesassociated with development of the fetus, so that one can identify theeffect of reduction in expression of the particular gene. Where the genemay be involved in regulation of a number of other genes, one can definethe effect of the absence of such gene on various aspects of thedevelopment of the fetus. The subject oligomers can be designed to bindto homeodomains, so that the transcription of one or more genes may beinhibited. In addition, one can use the subject compositions duringvarious periods during the development of the fetus to identify whetherthe gene is being expressed and what the effect is of the gene at theparticular stage of development.

With single cell organisms, one can determine the effect of the lack ofa particular expression product on the virulence of the organism, thedevelopment of the organism, the proliferation of the organism, and thelike. In this way, one can determine targets for drugs to inhibit thegrowth and infectiousness of the organism.

In an animal model, one can provide for inhibition of expression ofparticular genes, reversibly or irreversibly, by administering thesubject compositions to the host in a variety of ways, oral orparenteral, by injection, at a particular site where one wishes toinfluence the transcription, intravascularly, subcutaneously, or thelike. By inhibiting transcription, one can provide for a reversible“knock out,” where by providing for continuous intravenousadministration, one can greatly extend the period in which thetranscription of the gene is inhibited. Alternatively, one may use abolus of the subject oligomers and watch the effect on variousphysiological parameters as the bolus becomes dissipated. One canmonitor the decay of the effect of the inhibition, gaining insight intothe length of time the effect lasts, the physiological processesinvolved with the inhibition and the rate at which the normalphysiological response occurs. Instead, one can provide for covalentbonding of the oligomer to the target site, using alkylating agents,light activated bonding groups, intercalating groups, etc.

It is also possible to upregulate genes, by downregulating other genes.In those instances where one expression product inhibits the expressionof another expression product, by inhibiting the expression of the firstproduct, one can enhance the expression of the second product.Similarly, transcription factors involve a variety of cofactors to forma complex, one can enhance complex formation with one transcriptionfactor, as against another transcription factor, by inhibitingexpression of the other transcription factor. In this way one can changethe nature of the proteins being expressed, by changing the regulatoryenvironment in the cell.

The target sequence may be associated with the 5′-untranslated region,namely the transcriptional initiation region, an enhancer, which may bein the 5′-untranslated region, the coding sequence or introns, thecoding region, including introns and exons, the 3′-untranslated region,or distal from the gene.

The subject compositions may be presented as liposomes, being present ofthe lumen of the liposome, where the liposome may be combined withantibodies to surface membrane proteins or basement membrane proteins,ligands for cellular receptors, or other site directing compound, tolocalize the subject compositions to a particular target. See. forexample, Theresa and Mouse, Adv. Drug Delivery Rev. 1993, 21, 117-133;Huwyler and Partridge, Proc. Natl. Acad. Sci. USA 1996, 93, 11421-11425;Dzau et al., Proc. Natl. Acad. Sci. USA 1996, 93, 11421-11425; and Zhuet al., Science, 1993, 261, 209-211. The subject compositions may beadministered by catheter to localize the subject compositions to aparticular organ or target site in the host. Generally, theconcentration at the site of interest should be at least about 0.1 mM,e.g. intracellular or in the extracellular medium, preferably at leastabout 1 nM, usually not exceeding 1 mM, more usually not exceeding about100 nM. To achieve the desired intracellular concentration, theconcentration of the oligomers extracellularly will generally be greaterthan the desired intracellular concentration, ranging from about 2 to1000 times or greater the desired intracellular concentration. Ofcourse, where the toxicity profile allows for higher concentrations thanthose indicated for intracellular or extracellular concentrations, thehigher concentrations may be employed, and similarly, where theaffinities are high enough, and the effect can be achieved with lowerconcentrations, the lower concentrations may also be employed.

The subject compositions can be used to modulate physiological processesin vivo for a variety of reasons. In non-primates, particularly domesticanimals, in animal husbandry and breeding, one can affect thedevelopment of the animal by controlling the expression of particulargenes, modify physiological processes, such as accumulation of fat,growth, response to stimuli, etc. One can also use the subjectcompositions for therapeutic purposes in mammals. Domestic animalsinclude feline, murine, canine, lagomorpha, bovine, ovine, canine,porcine, etc.

The subject compositions may used therapeutically to inhibitproliferation of particular target cells, inhibit the expression of oneor more genes related to an indication, change the phenotype of cells,either endogenous or exogenous to the host, where the native phenotypeis detrimental to the host. Thus, by providing for binding tohousekeeping or other genes of bacteria or other pathogen, particularlygenes specific to the pathogen, one can provide for inhibition ofproliferation of the particular pathogen. Various techniques may be usedto enhance transport across the bacterial wall, such as various carriersor sequences, such as polylysine, poly(E-K), nuclear localizationsignal, cholesterol and cholesterol derivatives, liposomes, protamine,lipid anchored polyethylene glycol, phosphatides, such asdioleoxyphosphatidylethanolamine, phosphatidyl choline,phosphatidylglycerol, α-tocopherol, cyclosporin, etc. In many cases, thesubject compositions may be mixed with the carrier to form a dispersedcomposition and used as the dispersed composition. Similarly, where agene may be essential to proliferation or protect a cell from apoptosis,where such cell has undesired proliferation, the subject compositionscan be used to inhibit the proliferation by inhibiting transcription ofessential genes. This may find application in situations such ascancers, such as sarcomas, carcinomas and leukemias, restenosis,psoriasis, lymphopoiesis, atherosclerosis, pulmonary fibrosis, primarypulmonary hypertension, neurofibromatosis, acoustic neuroma, tuberoussclerosis, keloid, fibrocystic breast, polycystic ovary and kidney,scleroderma, rheumatoid arthritis, ankylosing spondilitis,myelodysplasia, cirrhosis, esophageal stricture, sclerosing cholangitis,retroperitoneal fibrosis, etc. Inhibition may be associated with one ormore specific growth factors, such as the families of platelet-derivedgrowth factors, epidermal growth factors, transforming growth factor,nerve growth factor, fibroblast growth factors, e.g. basic and acidic,keratinocyte fibroblast growth factor, tumor necrosis factors,interleukins, particularly interleukin 1, interferons, etc. In othersituations, one may wish to inhibit a specific gene which is associatedwith a disease state, such as mutant receptors associated with cancer,inhibition of the arachidonic cascade, inhibition of expression ofvarious oncogenes, including transcription factors, such as ras, myb,myc, sis, src, yes, fps/fes, erbA, erbB, ski, jun, crk, sea, rel, fms,abl, met, trk, mos, Rb-1 , etc. Other conditions of interest fortreatment with the subject compositions include inflammatory responses,skin graft rejection, allergic response, psychosis, sleep regulation,immune response, mucosal ulceration, withdrawal symptoms associated withtermination of substance use, pathogenesis of liver injury,cardiovascular processes, neuronal processes. Particularly, wherespecific T-cell receptors are associated with autoimmune diseases, suchas multiple sclerosis, diabetes, lupus erythematosus, myasthenia gravis,Hashimoto's disease, cytopenia, rheumatoid arthritis, etc., theexpression of the undesired T-cell receptors may be diminished, so as toinhibit the activity of the T-cells. In cases of reperfusion injury orother inflammatory insult, one may provide for inhibition of enzymesassociated with the production of various factors associated with theinflammatory state and/or septic shock, such as TNF, enzymes whichproduce singlet oxygen, such as peroxidases and superoxide dismutase,proteases, such as elastase, INFγ, IL-2, factors which induceproliferation of mast cells, eosinophils, IgG₁, IgE, regulatory T cells,etc., or modulate expression of adhesion molecules in leukocytes andendothelial cells.

Other opportunities for use of the subject compositions includemodulating levels of receptors, production of ligands, production ofenzymes, production of factors, reducing specific cell populations,changing phenotype and genotype of cells, particularly as associatedwith particular organs and tissues, modifying the response of cells todrugs or other stimuli, e.g. enhancing or diminishing the response,inhibiting one of two or more alleles, repressing expression of targetgenes, particularly as related to clinical studies, modification ofbehavior, modification of susceptibility to disease, response tostimuli, response to pathogens, response to drugs, therapeutic orsubstances of abuse, etc.

Individual compositions may be employed or combinations, directed to thesame dsDNA region, but different target sequences, contiguous or distal,or different DNA regions. Depending upon the number of genes which onewishes to target, the composition may have one or a plurality ofoligomers or pairs of oligomers which will be directed to differenttarget sites.

The subject compositions may be used as a sole therapeutic agent or incombination with other therapeutic agents. Depending upon the particularindication, other drugs may also be used, such as antibiotics, antisera,monoclonal antibodies, cytokines, anti-inflammatory drugs, and the like.The subject compositions may be used for acute situations or in chronicsituations, where a particular regimen is devised for the treatment ofthe patient. The compositions may be prepared in physiologicallyacceptable media and stored under conditions appropriate for theirstability. They may be prepared as powders, solutions or dispersions, inaqueous media, alkanols, e.g. ethanol and propylene glycol, inconjunction with various excipients, etc. The particular formulationwill depend upon the manner of administration, the desiredconcentration, ease of administration, storage stability, and the like.The concentration in the formulation will depend upon the number ofdoses to be administered, the activity of the oligomers, theconcentration required as a therapeutic dosage, and the like. Thesubject compositions may be administered orally, parenterally, e.g.intravenously, subcutaneously, intraperitoneally, transdermally, etc.The subject compositions may be formulated in accordance withconventional ways, associated with the mode of treatment. As a result ofthe formulation, the subject compositions are introduced into the cells,either as a directed introduction to a specific cell target or as randomintroduction into a number of different cell types. However, the subjectcompositions will only have an effect in those cells in which the targetdsDNA is being transcribed or there is some other mechanism whereby thebinding of the subject compositions can affect the mechanism. In thisway selectivity can be achieved, since the only productive result willbe in cells where the target dsDNA has an effect which is modified bythe binding of the subject compositions to the dsDNA.

The subject compounds may be prepared, conveniently employing a solidsupport. See, for example, Baird and Dervan, J. Am. Chem. Soc. 1996,118, 6141; PCT application, U.S. Ser. 97/03332. For solid phasesynthesis, the oligomer is grown on the solid phase attached to thesolid phase by a linkage which can be cleaved by a single step process.The addition of an aliphatic amino acid at the C-terminus of theoligomers allows the use of Boc-β-alanine-Pam-resin which iscommercially available in appropriate substitution levels (0.2 mmol/g).Aminolysis may be used for cleaving the polyamide from the support. Inthe case of the N-methyl 4-amino-2-carboxypyrrole and the N-methyl4-amino-2-carboxyimidazole, the activated esters such asN-hydroxysuccimidyl, 1,2,3-hydroxybenzotriazoyl, or the like may beemployed, with the amino groups protected by Boc or Fmoc, with themonomers added sequentially in accordance with conventional techniques.For further details, see the references cited in the related literature,which are incorporated herein by reference, as well as the Experimentalsection.

The following examples are offered by way of illustration, and not byway of limitation.

EXPERIMENTAL Example 1 Solid Phase Synthesis of Polyamides ContainingImidazole and Pyrrole Amino Acids

¹Baird & Dervan, J. Am. Chem. Soc., 1996,118, 6141.

Boc-β-alanine-(4-carboxamidomethyl)-benzyl-ester-copoly(styrene-divinylbenzene)resin (Boc-β-alanine-Pam-Resin), dicyclohexylcarbodiimide (DCC),hydroxybenzotriazole (HOBt),2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU), Boc-glycine, and Boc-β-alanine were purchased from PeptidesInternational. N,N-diisopropylethylamine (DIEA), N,N-dimethylformamide(DMP), N-methylpyrrolidone (NMP), and DMSO/NMP were purchased fromApplied Biosystems. Boc-γ-aminobutyric acid was from NOVA Biochem,dichloromethane (DCM) and triethylamine (TEA) was reagent grade from EM,thiophenol (PhSH), dimethylaminopropylamine, trichloroacetyl chloride,N-methylpyrrole, and N-methylimidazole from Aldrich, and trifluoroaceticacid (TFA) from Halocarbon. All reagents were used without furtherpurification.

MONOMER SYNTHESES 4-Nitro-2-trichloroacetMl-1-methylprrole

To a well stirred solution of trichloroacetyl chloride (1 kg, 5.5 mole)in 1.5 liter ethyl ether in a 12 liter flask was added dropwise over aperiod of 3 h a solution of N-methylpyrrole (0.45 kg, 5.5 mole) in 1.5liter anhydrous ethyl ether. The reaction was stirred for an additional3 hours and quenched by the dropwise addition of a solution of 400 gpotassium carbonate in 1.5 liters water. The layers were separated andthe ether layer concentrated in vacuo to provide2-(trichloroacetyl)pyrrole (1.2 kg, 5.1 mol) as a yellow crystallinesolid sufficiently pure to be used without further purification. To acooled (−40° C.) solution of 2-(trichloroacetyl) pyrrole (1.2 kg, 5.1mol) in acetic anhydride (6 L) in a 12 L flask equipped with amechanical stirrer was added 440 mL fuming nitric acid over a period of1 hour while maintaining a temperature of (−40° C.). The reaction wascarefully allowed to warm to room temperature and stir an additional 4h. The mixture was cooled to −30° C., and isopropyl alcohol (6 L) added.The solution was stirred at −20° C. for 30 min during which time a whiteprecipitate forms. The solution was allowed to stand for 15 min and theresulting precipitate collected by vacuum filtration.

Methyl 4-nitropyrrole-2-carboxylate

To a solution of 4-Nitro-2-trichloroacetyl-1-methylpyrrole (800 g, 2.9mol) in 2.5 L methanol in a 4 L Erlenmeyer flask equipped with amechanical stirrer was added dropwise a solution of NaH (60% dispersionin oil) (10 g, 0.25 mol) in 500 mL methanol. The reaction was stirred 2h at room temperature, and quenched by the addition of conc. sulfuricacid (25 mL). The reaction was then heated to reflux, allowed to slowlycool to room temperature. Product crystallized as white needles whichwere collected by vacuum filtration.

Methyl 4-amino-1-methyl-pyrrole-2-carboxylate Hydrochloride

Methyl-4-nitropyrrole-2-carboxylate 4 (450 g, 2.8 mol) was dissolved inethyl acetate (8 L). A slurry of 40 g of 10% Pd/C in 800 mL ethylacetate was then added and the mixture stirred under a slight positivepressure of hydrogen (c.a. 1.1 atm) for 48 h. Pd/C was removed byfiltration through celite, washed 1×50 mL ethyl acetate, and the volumeof the mixture reduced to c.a. 500 mL. 7 L of cold ethyl ether was addedand HCl gas gently bubbled through the mixture. The precipitated aminehydrochloride was then collected by vacuum filtration to yield a whitepowder (380 g, 81.6%).

4-[(tert-Butoxycarbonyl)amino]-1-methylpyIrole-2-carboxylic Acid

Methyl 4-amino-1-methyl-pyrrole-2-carboxylate hydrochloride (340 g, 1.8mol) was dissolved in 1 L of 10% aqueous sodium carbonate in a 3 L flaskequipped with a mechanical stirrer, di-t-butyldicarbonate (400 g, 2.0mmol) slurried in 500 mL of dioxane was added over a period of thirtymin., maintaining a temperature of 20° C. The reaction was allowed toproceed for three h and was determined complete by TLC, cooled to 5° C.for 2 h and the resulting white precipitate collected by vacuumfiltration. The Boc-pyrrole ester contaminated with Boc-anhydride wasdissolved in 700 mL MeOH, 700 mL of 2M NaOH was added and the solutionheated at 60° C. for 6 h. The reaction was cooled to room temperature,washed with ethyl ether (4×1000 mL), the pH of the aqueous layer reducedto c.a. 3 with 10% (v/v) H₂SO₄, and extracted with ethyl acetate (4×2000mL). The combined ethyl acetate extracts were dried (sodium sulfate) andconcentrated in vacuo to provide a tan foam. The foam was dissolved in500 mL of DCM and 2 L petroleum ether added, the resulting slurry wasconcentrated in vacuo. The reaction was redissolved and concentratedthree additional times to provide a fine white powder (320 g, 78%yield).

1,2,3-Benzotriazol-1-yl4-[(tert-butoxycarbonyl)-amino]-1-methylpyrrole-2-carboxylate

Boc-Py-acid (31 g, 129 mmol) was dissolved in 500 ML DMF, HOBt (17.4 g,129 mmol) was added followed by DCC (34 g, 129 mmol). The reaction wasstirred for 24 h and then filtered dropwise into a well stirred solutionof 5 L of ice water. The precipitate was allowed to sit for 15 min at 0°C. and then collected by filtration. The wet cake was dissolved in 500mL DCM, and the organic layer added slowly to a stirred solution of coldpetroleum ether (4° C.). The mixture was allowed to stand at −20° C. for4 h and then collected by vacuum filtration and dried in vacuo toprovide a finely divided white powder (39 g, 85% yield).

Ethyl 1-Methylimidazole-2-carboxylate

N-methylimidazole (320 g, 3.9 mol) was combined with 2 L acetonitrileand 1 L triethylamine in a 12 L flask equipped with a mechanical stirrerand the solution cooled to −20° C. Ethyl chloroformate (1000 g, 9.2 mol)was added with stirring, keeping the temperature between −20° C. and−25° C. The reaction was allowed to lowly warm to room temperature andstir for 36 h. Precipitated triethylamine hydrochloride was removed byfiltration and the solution concentrated in vacuo at 65° C. Theresulting oil was purified by distillation under reduced pressure (2torr, 102° C.) to provide a white solid (360 g, 82% yield).

Ethyl 1-Methyl-4-nitroimidazole-2-carboxylate

Ethyl 1-methylimidazole-2-carboxylate was carefully dissolved in 1000 mLof concentrated sulfuric acid cooled to 0° C. 90% nitric acid (1 L) wasslowly added maintaining a temperature of 0° C. The reaction was thenrefluxed with an efficient condenser (−20° C.) in a well ventilated hoodfor 50 min. The reaction was cooled with an ice bath, and quenched bypouring onto 10 L ice. The resulting blue solution was then extractedwith 20 L DCM, the combined extracts dried (sodium sulfate) andconcentrated in vacuo to yield a tan solid which was recrystallized from22 L of 21:1 carbon tetrachloride/ethanol. The resulting white crystalsare collected by vacuum filtration.

Ethyl 4-Amino-1-methylimidazole-2-carboxylate Hydrochloride

Ethyl 1-methyl-4-nitroimidazole-2-carboxylate (103 g, 520 mmol) wasdissolved in 5 L of 1:1 ethanol/ethyl acetate. 20 g 10% Pd/C slurried in500 mL ethyl acetate was added and the mixture stirred under a slightpositive pressure of hydrogen (c.a. 1.1 atm) for 48 h. The reactionmixture was filtered, concentrated in vacuo to a volume of 500 mL and 5L of cold anhydrous ethyl ether added. Addition of HCl gas provided awhite precipitate. The solution was cooled at −20° C. for 4 h and theprecipitate collected by vacuum filtration and dried in vacuo to provide(75 g, 78% yield) of a fine white powder.

4-[(tert-Butoxycarbonyl)amino]-1-methylimidazole-2-carboxylic Acid

Ethyl 4-amino-1-methylimidazole-2-carboxylate hydrochloride (75 g, 395mmol) was dissolved in 200 mL DMF. DIEA (45 mL, 491 mmol) was addedfollowed by di-t-butyldicarbonate (99 g, 491 mmol). The mixture wasshaken at 60° C. for 18 h, allowed to assume room temperature, andpartitioned between 500 mL brine, 500 mL ethyl ether. The ether layerwas extracted with (2×200 mL each) 10% citric acid, brine, satd. sodiumbicarbonate and brine, dried over sodium sulfate and concentrated invacuo to yield the Boc-ester contaminated with 20% Boc-anhydride asindicated by ¹H NMR. The Boc-ester, used without further purification,was dissolved in 200 mL 1 M NaOH. The reaction mixture was allowed tostand for 3 h at 60° C. with occasional agitation. The reaction mixturewas cooled to 0° C., and carefully neutralized with 1 M HCl to pH 2, atwhich time a white gel formed. The gel was collected by vacuumfiltration, frozen before drying, and remaining water lyophilized toyield a white powder.

4-[(tert-Butoxycarbonyl)amino]-1-methylpyrrole-2-(4-carboxamide-methylimidazole)-2-carboxylicAcid

This compound was prepared as described below for(-[(tert-Butoxycarbonyl)amino]-butyricacid-(4-carboxamido-1-methyl-imidazole)-2-carboxylic acid, substitutingBoc-Pyrrole acid for Boc-γ-aminobutyric acid. (4.1 g, 91% yield).

(γ-[(tert-Butoxycarbonyl)amino]-butyricAcid-(4-carboxamido-1-methyl-imidazole)-2-carboxylic Acid

To a solution of Boc-(-aminobutyric acid (10 g, 49 mmol) in 40 mL DMFwas added 1.2 eq HOBt (7.9 g, 59 mmol) followed by 1.2 eq DCC (11.9 g,59 mmol). The solution was stirred for 24 h, and the DCU removed byfiltration. Separately, to a solution of ethyl4-nitro-1-methylimidazole-2-carboxylate (9.8 g, 49 mmol) in 20 mL DMFwas added Pd/C catalyst (10%, 1 g), and the mixture was hydrogenated ina Parr bomb apparatus (500 psi H2) for 2 h. The catalyst was removed byfiltration through celite and filtrate immediately added to the HOBtester solution. An excess of DIEA (15 mL) was then added and thereaction stirred at 37° C. for 48 h. The reaction mixture was then addeddropwise to a stirred solution of ice water and the resultingprecipitate collected by vacuum filtration to provide crude ethyl4-[[[3-[(tert-butoxycarbonyl]amino]propyl]carbonylamino]-1-methylimidazole-2-carboxylate(5 g, 14.1 mmol). To the crude ester dissolved in 50 mL methanol wasadded 50 mL 1M KOH and the resulting mixture allowed to stir for 6 h at37° C. Excess methanol was removed in vacuo and the resulting solutionacidified by the addition of 1 M HCl. The resulting precipitate wascollected by vacuum filtration and dried in vacuo to yield a brownpowder. (4.4 g, 89% yield).

Solid Phase Syntheses

Activation of Imidazole-2-carboxylic acid, (γ-aminobutyric acid,Boc-glycine, and Boc-β-alanine. The appropriate amino acid or acid (2mmol) was dissolved in 2 mL DMF. HBTU (720 mg, 1.9 mmol) was addedfollowed by DIEA (1 mL) and the solution lightly shaken for at least 5min.

Activation of Boc-Imidazole Acid

Boc-imidazole acid (257 mg, 1 mmol) and HOBt (135 mg, 1 mmol) weredissolved in 2 mL DMF, DCC (202 mg, 1 mmol) is then added and thesolution allowed to stand for at least 5 min.

Activation of Boc-γ-Imidazole Acid and Boc-Pyrrole-Imidazole Acid

The appropriate dimer (1 mmol) and HBTU (378 mg, 1 mmol) are combined in2 mL DMF. DIEA (1 mL) is then added and the reaction mixture allowed tostand for 5 min.

Activation of Boc-Pyrrole Acid (for Coupling to Imidazole Amine)

Boc-Pyrrole acid (514 mg, 2 mmol) was dissolved in 2 mL dichloromethane,DCC (420 mg, 2 mmol) added, and the solution allowed to stand for 10min, DMAP (101 mg, 1 mmol) was added and the solution allowed to standfor 1 min.

Acetylation Mix

2 mL DMF, DIEA (710 μL, 4.0 mmol), and acetic anhydride (380 μL, 4.0mmol) were combined immediately before use.

Manual Synthesis Protocol

Boc-β-alanine-Pam-Resin (1.25 g, 0.25 mmol) is placed in a 20 mL glassreaction vessel, shaken in DMF for 5 min and the reaction vesseldrained. The resin was washed with DCM (2×30 s.) and the Boc groupremoved with 80% TFA/DCM/0.5 M PhSH, 1×30 s., 1×20 min. The resin waswashed with DCM (2×30 s.) followed by DMF (1×30 s.). A resin sample(5-10 mg) was taken for analysis. The vessel was drained completely andactivated monomer added, followed by DIEA if necessary. The reactionvessel was shaken vigorously to make a slurry. The coupling was allowedto proceed for 45 min, and a resin sample taken. The reaction vessel wasthen washed with DCM, followed by DMF.

Machine-Assisted Protocols

Machine-assisted synthesis was performed on a ABI 430A synthesizer on a0.18 mmol scale (900 mg resin; 0.2 mmol/gram). Each cycle of amino acidaddition involved: deprotection with approximately 80% TFA/DCM/0.4 MPhSH for 3 minutes, draining the reaction vessel, and then deprotectionfor 17 minutes; 2 dichloromethane flow washes; an NMP flow wash;draining the reaction vessel; coupling for 1 hour with in situneutralization, addition of dimethyl sulfoxide (DMSO)/NMP, coupling for30 minutes, addition of DIEA, coupling for 30 minutes; draining thereaction vessel; washing with DCM, taking a resin sample for evaluationof the progress of the synthesis by HPLC analysis; capping with aceticanhydride/DIEA in DCM for 6 minutes; and washing with DCM. A doublecouple cycle is employed when coupling aliphatic amino acids toimidazole, all other couplings are performed with single couple cycles.

The ABI 430A synthesizer was left in the standard hardware configurationfor NMP-HOBt protocols. Reagent positions 1 and 7 were DIEA, reagentposition 2 was TFA/0.5 M thiophenol, reagent position 3 was 70%ethanolamine/methanol, reagent position 4 was acetic anhydride, reagentposition 5 was DMSO/NMP, reagent position 6 was methanol, and reagentposition 8 was DMF. New activator functions were written, one for directtransfer of the cartridge contents to the concentrator (switch list 21,25, 26, 35, 37, 44), and a second for transfer of reagent position 8directly to the cartridge (switch list 37, 39, 45, 46).

Boc-Py-OBt ester (357 mg, 1 mmol) was dissolved in 2 mL DMF and filteredinto a synthesis cartridge. Boc-Im acid monomer was activated(DCC/HOBt), filtered, and placed in a synthesis cartridge.Imidazole-2-carboxylic acid was added manually. At the initiation of thecoupling cycle the synthesis was interrupted, the reaction vessel ventedand the activated monomer added directly to the reaction vessel throughthe resin sampling loop via syringe. When manual addition was necessaryan empty synthesis cartridge was used. Aliphatic amino acids (2 mmol)and HBTU (1.9 mmol) were placed in a synthesis cartridge. 3 mL of DMFwas added using a calibrated delivery loop from reagent bottle 8,followed by calibrated delivery of 1 mL DIEA from reagent bottle 7, anda 3 minute mixing of the cartridge.

The activator cycle was written to transfer activated monomer directlyfrom the cartridge to the concentrator vessel, bypassing the activatorvessel. After transfer, 1 mL of DIEA was measured into the cartridgeusing a calibrated delivery loop, and the DIEA solution combined withthe activated monomer solution in the concentrator vessel. The activatedester in 2:1 DMF/DIEA was then transferred to the reaction vessel. Alllines were emptied with argon before and after solution transfers.

ImPyPy-γ-PyPyPy-β-alanine-Dp

ImPyPy-γ-PyPyPy-β-alanine-Pam-Resin was prepared by machine-assistedsynthesis protocols. A sample of resin (1 g, 0.17 mmol) was placed in a20 mL glass scintillation vial, 4 mL of dimethylaminopropylamine added,and the solution heated at 55° C. for 18 h. Resin is removed byfiltration through a disposable propylene filter and 16 mL of wateradded. The polyamide/amine mixture was purified directly by preparatoryHPLC and the appropriate fractions lyophilized to yield a white powder.

Stepwise HPLC Analysis

A resin sample (c.a. 4 mg) was placed in a 4 mL glass test tube, 200 μLof N,N-dimethylaminopropylamine was added and the mixture heated at 100°C. for 5 min. The cleavage mixture was filtered and a 25 μL sampleanalyzed by analytical HPLC at 254 nm.

Example 2 β-Alanine and γ-Aminobutyric Acid are Turn and OverlappedSpecific “Guide” Amino Acids Which may be Combined Predictably Withinthe Same Molecule.²

²Tranger et al, Chemistry and Biology, 1996, 3, 369-377.

Synthesis of Polyamides ImPyPy-γ-aminobutrric Acid-ImPyPy-β-alanine-Dpand ImPyPy-γ-aminobutyric Acid-ImPyPy-β-alanine-PyPyPy-G-Dp

All polyamides were prepared in high purity using solid phase syntheticmethodology as described above. Polyamides were assembled in a stepwisemanner on Boc-β-alanine-Pam resin and Boc-glycine-Pam-resinrespectively. Polyamides ImPyPy-γ-aminobutyric acid-ImIyPy-β-alanine-Dp,ImPyPy-γ-aminobutyric acid-ImPyPy-β-alanine-PyPyPy-G-Dp andImPyPy-γ-aminobutyric acid-ImPyPy-β-alanine-PyPyPy-G-Dp-NH₂ were cleavedfrom the support with an appropriate primary amine and purified byreversed-phase HPLC to provide 10-30 mg of polyamide. A primary aminegroup suitable for post-synthetic modification can be provided. Aminemodified polyamides are treated with an excess of the dianhydride ofEDTA, unreacted anhydride hydrolyzed, and the EDTA modified polyamideImPyPy-γ-aminobutyric acid-ImPyPy-β-alanine-PyPyPy-G-Dp-EDTA isolated byreversed-phase HPLC.

ImPyPy-γ-aminobutyric Acid-ImPyPy-β-alanine-PyPyPy-G-Dp-NH₂

Polyamide was prepared by machine-assisted solid phase methods as awhite powder (29 mg, 59% recovery).

ImPyPy-γ-aminobutyric Acid-ImPyPy-β-alanine-PyPyPy-G-Dn-EDTA

EDTA-dianhydride (50 mg) was dissolved in 1 mL DMSO/NMP solution and 1mL DIEA by heating at 55° C. for 5 min. The dianhydride solution wasadded to ImPyPy-γ-aminobutyric acid-ImPyPy-β-alanine-PyPyPy-G-Dp-NH₂(9.0 mg, 5 μmol) dissolved in 750 μL DMSO. The mixture was heated at 55°C. for 25 min, treated with 3 mL 0.1 M NaOH, and heated at 55° C. for 10min. 0.1% TFA was added to adjust the total volume to 8 mL and thesolution purified directly by reversed-phase HPLC to provideImPyPy-γ-aminobutyric acid-ImPyPy-βalanine-PyPyPy-G-Dp-EDTA as a whitepowder (3 mg, 30% recovery after HPLC purification).

Preparation of ³²P-labeled DNA

Plasmid pJT8 was prepared by hybridizing two sets of 5′-phosphorylatedcomplementary oligonucleotides,5′-CCGGGAACGTAGCGTACCGGTCGCAAAAAGACAGGCTCGA-3′ (SEQ ID NO:3), and5′-GGCGTCGAGCCTGTCTTTTTGCGACCGGTACGCTACGTTC-3′ (SEQ ID NO:4), and5′-CGCCGCATATAGACAGGCCCAGCTGCGTCCTAGCTAGCGTCGTAGCGTCTTAAGAG-3′ (SEQ IDNO:5) and 5′-TCGACTCTTAAGACGCTACGACGCTAGCTAGGACGCAGCTGGGCCTGTCTATATGC-3′(SEQ ID NO:6), and ligating the resulting duplexes to the large pUC19AvaI/SalI restriction fragment. The 3′-³²p end-labeled AflII/FspIfragment was prepared by digesting the plasmid with AflII andsimultaneously filling in using Sequenase,[″α-³²P]-deoxyadenosine-5′-triphosphate, and[″α-³²P]-thymidine-5′-triphosphate, digesting with FspI, and isolatingthe 247 bp fragment by nondenaturing gel electrophoresis. The5′-³²P-end-labeled AflII/FspI fragment was prepared using standardmethods. A and G sequencing were carried out as described.³ Standardmethods were used for all DNA manipulations.⁴

Affinity Cleavage Reactions

³Maxam & Gilbert, Methods Enzymol., 1980, 65, 499-560; Iverson & Dervan,Methods Enzymol., 1987, 15, 7823-7830; Sambrook et al, 1989, MolecularCloning, 2nd ed., Cold Spring Harbor Laboratory Press:Cold SpringHarbor, N.Y.

⁴Sambrook et al, 1989, Molecular Cloning, 2nd ed., Cold Spring HarborLaboratory Press: Cold Spring Harbor, N.Y.

All reactions were executed in a total volume of 400 mL. A stocksolution of EDTA-modified polyamide or H20 was added to a solutioncontaining labeled restriction fragment (15,000 cpm), affording finalsolution conditions of 20 mM HEPES, 200 mM NaCl, 50 μg/mL glycogen, andpH 7.3. Subsequently, 20 μL of freshly prepared 20 mM Fe(NH₄)₂(SO₄)₂ wasadded and the solution allowed to equilibrate for 20 min. Cleavagereactions were initiated by the addition of 40 μ of 50 mMdithiothreitol, allowed to proceed for 12 min at 22° C., then stopped bythe addition of 1 mL of ethanol. Reactions were precipitated and thecleavage products separated using standard methods. Next, 10 μL of asolution containing calf thymus DNA (140 μM base-pair) (Pharmacia) andglycogen (2.8 mg/mL) was added, and the DNA precipitated. The reactionswere resuspended in 1×TBE/80% formamide loading buffer, denatured byheating at 85° C. for 10 min, and placed on ice. The reaction productswere separated by electrophoresis on an 8% polyacrylamide gel (5%crosslink, 7 M urea) in 1×TBE at 2000 V. Gels were dried and exposed toa storage phosphor screen. Relative cleavage intensities were determinedby volume integration of individual cleavage bands using ImageQuantsoftware.

Quantitative DNase I Footprint Titration Experiments

All reactions were executed in a total volume of 400 μL. A polyamidestock solution or H₂O (for reference lanes) was added to an assay buffercontaining radiolabeled restriction fragment (15,000 cpm), affordingfinal solution conditions of 10 mM TrisHCl, 10 mM KCl, 10 mM MgCl₂, 5 mMCaCl₂, pH 7.0, and either (i) 1 pM-10 nM polyamide or (ii) no polyamide(for reference lanes). The solutions were allowed to equilibrate at 22°C. for (i) 12 h for polyamide 1 or (ii) 36 h for polyamide 2.Footprinting reactions were initiated by the addition of 10 mL of aDNase I stock solution (at the appropriate concentration to give 55%intact DNA) containing 1 mM dithiothreitol and allowed to proceed forseven min at 22° C. The reactions were stopped by the addition of 50 μLof a solution containing 2.25 M NaCl, 150 mM EDTA, 0.6 mg/mL glycogen,and 30 gM base-pair calf thymus DNA, and ethanol precipitated. Reactionswere resuspended in 1×TBE/80% formamide loading buffer, denatured byheating at 85° C. for 10 min, and placed on ice. The reaction productswere separated by electrophoresis on an 8% polyacrylamide gel (5%crosslink, 7 M urea) in 1×TBE at 2000 V. Gels were dried and exposed toa storage phosphor screen (Molecular Dynamics).

Quantitation and Data Analysis

Data from the footprint titration gels were obtained using a MolecularDynamics 400S PhosphorImager followed by quantitation using ImageQuantsoftware (Molecular Dynamics). Background-corrected volume integrationof rectangles encompassing the footprint sites and a reference site atwhich DNase I reactivity was invariant across the titration generatedvalues for the site intensities (I_(site)) and the reference intensity(I_(ref)). The apparent fractional occupancy (2_(app)) of the sites werecalculated using the equation (1): $\begin{matrix}{Q_{app} = {1 - \frac{I_{site}/I_{ref}}{I_{site}^{o}/I_{ref}^{o}}}} & (1)\end{matrix}$

where I°_(site) and I°_(ref) are the site and reference intensities,respectively, from a control lane to which no polyamide was added. The([L]_(tot), Q_(app)) data points were fit to a general Hill equation (eq2) by minimizing the difference between Q_(app) and Q_(fit):$\begin{matrix}{Q_{fit} = {Q_{\min} + {\left( {Q_{\max} - Q_{\min}} \right)\quad \frac{{K_{a}^{n}\lbrack L\rbrack}_{tot}^{n}}{1 + {K_{a}^{n}\lbrack L\rbrack}_{tot}^{n}}}}} & (2)\end{matrix}$

where [L]_(tot) is the total polyamide concentration, K_(a) is theequilibrium association constant, and Q_(min) and Q_(max) are theexperimentally determined site saturation values when the site isunoccupied or saturated, respectively. The data were fit using anonlinear least-squares fitting procedure with K_(a), Q_(max), andQ_(min) as the adjustable parameters. For polyamideImPyPy-γ-aminobutyric acid-ImPyPy-β-alanine-Dp, binding isotherms forthe 5′-AGACA-3′ (SEQ ID NO: 11) target sites were adequately fit byLangmuir isotherms (eq 2, n=1), consistent with formation of 1:1polyamide-DNA complexes. For ImPyPy-γ-aminobutyricacid-ImPyPy-β-alanine-PyPyPy-G-Dp, steeper binding isotherms (eq 2,n=1.8-2.2) were observed at the target sites 5′-AAAAAGACA-3′ (SEQ ID NO:7) and 5′-ATATAGACA-3′ (SEQ ID NO: 8). The steepness of these isothermsmay be due to the very high equilibrium association constants at thesesites. Treatment of the data in this manner does not represent anattempt to model a binding mechanism. The data is a comparison of valuesof the apparent first-order association constant, a value thatrepresents the concentration of ligand at which a site ishalf-saturated. The binding isotherms were normalized using thefollowing equation: $\begin{matrix}{Q_{norm} = \frac{Q_{app} - Q_{\min}}{Q_{\max} - Q_{\min}}} & (3)\end{matrix}$

Four sets of data were used in determining each association constant.The method for determining association constants used here involves theassumption that [L]_(tot). [L]_(free), where [L]_(free) is theconcentration of polyamide free in solution (unbound). For very highassociation constants this assumption becomes invalid, resulting inunderestimated association constants. In the experiments described here,the DNA concentration is estimated to be ⁻⁵ pM. As a consequence,apparent association constants greater than ⁻ ¹⁰ M⁻¹.

DNA-Binding Orientation

⁵Wade et al, J. Am. Chem. Soc., 1992, 114, 8783-8794; Schultz et al, J.Am. Chem. Soc., 1982,104, 6861-6863.

Affinity cleavage⁵ experiments with ImPyPy-γ-aminobutyricacid-ImPyPy-β-alanine-PyPyPy-G-Dp-EDTAFe(II) (2-Fe(II)) on the 5′- or3′-³²P end-labeled 247 bp pJT4 AflII/FspI restriction fragment revealedthat this polyamide selectively binds the 5′-AAAAAGACA-3′ (SEQ ID NO: 7)and 5′-ATATAGACA-3′ (SEQ ID NO: 8) target sequences at subnanomolarconcentration. A single 3′-shifted cleavage pattern is observed at each9 bp site indicating that the polyamide is bound in one orientation withthe C-terminus at the 5′ end of the 5′-AAAAAGACA-3′ (SEQ ID NO: 7) and5′-ATATAGACA-3′ (SEQ ID NO: 8) sequences.

DNA-binding Affinity and Specificity

⁶Hertzberg & Dervan, J. Am. Chem. Soc., 1982, 104, 313-315.

⁷Fox & Waring, Nucleic Acids Res., 1984, 12, 9271-9285; Brenowitz et al,Methods Enzymol., 1986, 130, 132-181; Brenowitz, et al, Proc. Natl.Acad. Sci. U.S.A., 1986, 83, 8462-8466.

The exact locations and sizes of all binding sites were determined firstby preliminary MPEFe(II) footprinting experiments.⁶ Quantitative DNase Ifootprint titration experiments⁷ on the 3′-³²P-labeled 247 bprestriction fragment (10 mM TrisHCl, 10 mM KCl, 10 mM MgCl₂, 5 mM CaCl₂,pH 7.0, 22° C.) reveal that ImPyPy-γ-aminobutyricacid-ImPyPy-β-alanine-PyPyPy-G-Dp specifically binds 5′-AAAAAGACA-3′(SEQ ID NO: 7) and 5′-ATATAGACA-3′ (SEQ ID NO: 8) with equilibriumassociation constants of Ka=2×10¹⁰ M⁻¹ and Ka=8×10⁹ M⁻¹, respectively.Additional sites on the restriction fragment are bound with loweraffinity. For comparison, the six-ring hairpin polyamideImPyPy-γ-aminobutyric acid-ImPyPy-β-alanine-Dp binds 5′-aaaaAGACA-3′(SEQ ID NO: 7) and 5′-atatAGACA-3′ (SEQ ID NO: 8) with associationconstants of Ka=5×10⁷ M⁻¹ and Ka=9×10⁷ M⁻¹, respectively.

Relative to the six-ring polyamide ImPyPy-γ-aminobutyricacid-ImPyPy-β-alanine-Dp, the nine-ring polyamide ImPyPy-γ-aminobutyricacid-ImPyPy-β-alanine-PyPyPy-G-Dp binds 5′-AAAAAGACA-3′ (SEQ ID NO: 7)and 5′-ATATAGACA-3′ (SEQ ID NO: 8) with 400-fold and 100-fold higheraffinity, respectively. Similar binding enhancements have recently beenreported in a separate system.⁸ Addition of a C-terminal PyPyPy subunitusing a β-alanine linker is an effective strategy for increasing theDNA-binding affinity of hairpin polyamides that bind adjacent to an(A,T)₄ sequence.

⁸Trauger et al., Nature, 1996, 382, 559-561

Polyamide ImPyPy-γ-aminobutyric acid-ImPyPy-β-alanine-PyPyPy-G-Dp bindsseveral mismatch sites present on the 247 bp restriction fragment withhigh affinity. The two highest affinity mismatch sites, 5′-GAATTCACT-3′(SEQ ID NO: 9) (K_(a)=4.5×10⁹ M⁻¹) and 5′-GTTTTCCCA-3′ (SEQ ID NO: 10)(K_(a)=2.5×10⁹ M⁻¹), are bound with at least 5-fold reduced affinityrelative to the optimal match site 5′-AAAAAGACA-3′ (SEQ ID NO: 7)(formally mismatched base-pairs are highlighted), although this valuemay be a lower limit due to the uncertainty in the very high equilibriumassociation constant for the optimal match site. In contrast, thesix-ring polyamide ImPyPy-γ-aminobutyric acid-ImPyPy-β-alanine-Dp bindsmore strongly to the match site 5′-AGACA-3′ (SEQ ID NO: 11) over thesingle base-pair mismatch sites 5′-ATTCA-3′ (SEQ ID NO: 12) and5′-TTACA-3′ (SEQ ID NO: 13) by a factor of 10.

Example 3 Subnanomolar Lipand Binding with Single MismatchDifferentiation

⁹Baird and Dervan, J.Am. Chem. Soc.,1996, 118, 6141-6146

The DNA-binding affinities were evaluated of two eight-ring hairpinpolyamides, ImPyPyPy-γ-ImPyPyPy-β-Dp (1) and ImPyPyPy-γ-PyPyPyPy-β-Dp(2), which differ by a single amino acid, for two 6 base pair (bp)target sites, 5′-AGTACT-3′ (SEQ ID NO: 1) and 5′-AGTATT-3′ (SEQ ID NO:2), which differ by a single base pair. Based on the pairing rules forpolyamide-DNA complexes, the sites 5′-AGTACA-3′ (SEQ ID NO: 14) and5′-AGTATT-3′ (SEQ ID NO: 2) are for polyamide 1 “match” and “single basepair mismatch” sites, respectively, and for polyamide 2 “single basepair mismatch” and “match” sites, respectively (FIG. 2).

Polyamides 1 and 2 were synthesized by solid phase methods and purifiedby reversed phase HPLC⁹. The identity and purity of the polyamides wasverified by ¹H NMR, MALDI-TOF MS, and analytical HPLC. MALDI-TOF MS: 1,1223.4 (1223.3 calculated for M+H); 2, 1222.3 (1222.3 calculated forM+H). Equilibrium association constants for complexes of 1 and 2 withmatch and mismatch six base pair binding sites on a 3′-³²P-labeled 229bp restriction fragment were determined by quantitative DNase Ifootprint titration experiments¹⁰ (Table I ).

¹⁰Galas & Schmitz, Nucleic Acids Res., 1978, 5, 3157-3170; Fox & Waring,ibid, 1984, 19271-9285; Brenowitz et al., Meth. Enzym. 1986, 130,132-181

TABLE 1 Equilibrium association constants (M⁻¹) Binding Site 1 25′-ttAGTACTtg-3′ 3.7 × 10¹⁰ (0.8) 5.0 × 10⁸ (0.5) 5′-ttAGTATTtg-3′ 4.1 ×10⁸ (0.5) 3.5 × 10⁹ (0.8) The reported association constants are theaverage values obtained from three DNase 1 footprint titrationexperiments. The standard deviation for each data set is indicated inparentheses. Assays were carried out in the presence of 10 mM Tris.HCl,10 mM KCl, 10 mM MgCl₂, and 5 mM CaCl₂ at pH 7.0 and 22° C. The sixbase-pair binding sites are in capital letters, with flanking sequencesin lower-case letters.

Polyamide 1 binds its match site 5′AGTACT3′ (SEQ ID NO: 1) at 0.03 nMconcentration and its single base pair mismatch site 5′-AGTATT-3′ (SEQID NO: 2) with nearly 100-fold lower affinity. Polyamide 2 binds itsdesignated match site 5′-AGTATT-3′ (SEQ ID NO: 2) at 0.3 nMconcentration and its single base pair mismatch site 5′-AGTACT-3′ (SEQID NO: 1) with nearly 10-fold lower affinity. The specificity of 1 and 2for their respective match sites results from very small structuralchanges. Replacing a single nitrogen atom in 1 with C-H (as in 2)reduces the affinity of the polyamide. 5′-AGTACT-3′ (SEQ ID NO: 1)complex by ⁻75-fold representing a free energy difference of ⁻2.5kcal/mole. Similarly, replacing a C-H in 2 with N (as in 1) reduces theaffinity of the polyamidee.5′-AGTATT-3′ (SEQ ID NO: 2) complex ⁻10-fold,a loss in binding energy of 1.3 kcal/mol.

Quantitative DNase I footprint titration experiments with polyamides 1and 2 on the 3′-³²P-labeled 229 bp pJT8 AflII/FspI restriction fragment.Comparison lanes were A and G sequencing lanes; DNase I digestionproducts obtained in the absence of polyamide; DNase I digestionproducts obtained in the presence of 1 pM, 2 pM, 5 pM, 10 pM, 15 pM, 25pM, 40 pM, 65 pM, 0.1 nM, 0.15 nM, 0.25 nM, 0.4 nM, 0.65 nM, 1 nM, 2 nM,5 nM, and 10 nM polyamide, respectively; and intact DNA. Polyamidebinding sites for which association constants were determined for5′-AGTACT-3′ (SEQ ID NO: 1) and 5′-AGTATT-3′ (SEQ ID NO: 2). Additionalsites not analyzed were 5′-TGTAAA-3′ (SEQ ID NO: 15), 5′-TGTGCT-3′ (SEQID NO: 16), and 5′-TAAGTT-3′ (SEQ ID NO: 17). All reactions wereexecuted in a total volume of 400 μL. A polyamide stock solution or H₂Owas added to an assay buffer containing radiolabeled restrictionfragment, affording final solution conditions of 10 mM Tris.HCl, 10 mMKCl, 10 mM MgCl₂, 5 mM CaCl₂, and pH 7.0. The solutions were allowed toequilibrate for 12-15 h at 22° C. prior to initiation of footprintingreactions. Footprinting reactions, separation of cleavage products, anddata analysis were carried out as described elsewhere¹¹. Plasmid pJT8was prepared by hybridizing two 5′-phosphorylated complementaryoligonucleotides,

¹³Mrkisch et al., J. Am. Chem. Soc., 1994, 116, 7983-79885′-CCGGTTAGTATTTGGATGGGCCTGGTTAGTA-CTTGGATGGGAGACCGCCTGGGAATACCAGGTGTCGTATCTTAAGAG-3′(SEQID NO: 18) and5′-TCGACTCTTAAGATACGACACCTGGTATTCCCAGGCGGTCTCCCATCCAA-GTACTAACCAGGCCCATCCAAATACTAA-3′(SEQ ID NO: 19), and ligating the resulting duplex to the large pUC19AvaI/SalI restriction fragment.

Example 4 Intracellular Binding and Transcription Inhibition Methods

Polyamides. Polyamides were synthesized by solid phase methods.¹² Theidentity and purity of the polyamides was verified by ¹H NMR, matrixassisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS), and anlaytical HPLC. MALDI-TOF-MS: 1, 1223.4(1223.3-calcd for M+H); 2, 1222.3 (1222.3 calcd for M+H); 3, 1223.1(1223.3 calcd for M+H).

¹²Baird, E. E. and Dervan, P. B., J. Am. Chem. Soc., 1996, 118,6141-6146

Transcription inhibition in vitro. A high speed cytosolic extract fromunfertilized Xenopus egges was prepared as decribed.¹³ DNA templates fortranscription were the somatic-type 5S RNA gene contained in plasmidpX1s11¹⁴ (50 ng per reaction) and the tyrD tRNA gene contained inplasmid pTyrD¹⁵ (100 ng plasmid DNA per reaction), both from X. laevis.Transcription reactions (20 μL fmal volume) contained the followingcomponents: 2.5 μL extract. 9 ng (12 nM) of TFIIIA isolated fromimmature oocytes¹⁶, 0.6 mM ATP, UTP, CTP,0.02 mM GTP and 10 μCi of[α-³²P] GTP and the fmal buffer components 12 mM HEPES (pH 7.5), 60 mMKCl, 6 mM MgCl₂, 25 μM ZnCl₂, and 8% (v/v) glycerol. Plasmid DNAs werepre-incubated with polyamides in the same buffer prior to adding TFIIIAand other reaction components. RNA was pruified and analyzed on adenaturing 6% polyacrylamide gel. A Molecular Dynamics Phosphorimagerequipped with ImageQuant software was used to quantify the effect of thepolyamides on 5S and tRNA gene transcription.

¹³Hartl, P. et al., J. Cell Biol., 1993, 120, 613-624

¹⁴Peterson, R. C. et al., Cell, 1980, 20, 131-144

¹⁵Stutz, F. et al., Genes Dev., 1989, 3, 1190-1198

¹⁶Smith, D. R. et al., Cell, 1984, 37, 645-652

Transcription inhibition in vivo. Fibroblasts from a Xenopus kidneyderived cell line (kindly provided by Dr. P. Labhart, Scripps) weregrown at ambient temperature in 25 cm² culture flasks in Dulbecco'smodified Eagle medium containing 10% (v/v) fetal calf serum. Cells werepassaged for a minimum of three days prior to the addition of polyamideto the culture medium. Incubations were continued for various times andnuclei were prepared by hypotonic lysis and used as templates fortranscription as described.¹⁷ DNA content was determined by measuringthe absorbance of an aliquot of the isolated nuclei in 1% (w/v) sodiumdodecyl sulfate (using an extinction coefficient at 260 nM of 1 AU=50μg/mL DNA). The buffer components and labeled and unlabeled nucleosidetriphosphates were as for the plasmid transcription reactions. Reactionswere supplemented with 2 μL of RNA polymerase III (at approximately 50μg/mL)isolated from Xenopus oocytes.¹⁸

¹⁷Schlissel, M. S. and Brown, D. D., Cell, 1984, 37, 903-913

¹⁸Roeder, R. G., J. Biol. Chem., 1984, 258, 1932-1941

Results

The effect of polyamide 1 (ImPyPyPy-γ-ImPyPyPy-β-Dp) on TFIIIA bindingto a restriction fragment isolated from a 5S RNA gene-containing plasmidwas examinded. Zf1-3, a recombinant TFIIIA analog missing fmgers 4-9,binds in the major groove of the C-block promoter element (see FIG. 1).DNase I footprinting demonstrates that zfi-3 and polyamide 1 canco-occupy the same DNA molecule. When 5 nM polyamide 1 was preincubatedwith the same DNA target, the binding of nine finger TFIIIA wasinhibited by >90%. The differential inhibition of zf1-3 and full-lengthTFIIIA provides evidence that finger 4 interacts with or is placed inthe minor groove. Polyamide 1 does not inhibit TFIIA binding to 5S RNA.

Transcription of the 5S RNA gene in an in vitro system was monitored inthe presence of increasing concentrations (10-60 nM) of polyamide 1. Inthese experiments, polyamide 1 was added to a 5S RNA gene containingplasmid prior to the addition of exogenous TFIIIA (12 nM) and a crudeextract derived from unfertilized Xenopus eggs. As a control, a tyrosinetRNA gene was included on a separate plasmid in these reactions. ThetRNA gene has an upstream binding site for 1, but lacks a predictedprotein-polyamide interaction. Both genes are actively transcribed inthis system, either individually or in mixed template reactions.

Addition of 60 nM polyamide 1 inhibits 5S gene transcription by >80%.Only a small degree of non-specific inhibition of tRNA transcription isobserved at the concentrations of polyamide 1 required for efficient 5SRNA inhibition. The targeted 5S RNA gene is inhibited approximately10-fold more effectively than the control tRNA gene. Mismatch polyamides2 (ImPyPyPy-γ-PyPyPyPy-β-Dp) and 3 (ImPyImPy-β-PyPyPyPy-β-Dp) do notinhibit 5S RNA transcription at concentrations up to 60 nM. If theTFIIIA-DNA complex is first allowed to form, 30 nM polyamide 1 added,and the mixture incubated for 90 minutes prior to adding egg extract,efficient inhibition (80%) of 5S RNA transcription is also observed.Shorter incubation times result in less inhibition. The requiredincubation time of 90 minutes is similar to the measured half-life ofthe TFIIIA-DNA complex and supports that polyamide 1 forms a more stablecomplex with DNA than does TFIIIA.

The effect of the polyamides on 5S gene transcription in vivo wasmonitored. Xenopus kidney-derived fibroblasts were grown in the presenceof increasing concentrations of polyamide 1 in the culture medium forvarious times. We found that concentrations of polyamide up to 1 μM werenot toxic, as measured by cell density, if growth was limited to lessthan 72 hours. Nuclei were prepared from cells by hypotonic lysis andequivalent amounts of the isolated nuclei from control and treated cellswere used as templates for transcription with exogenous RNA polymeraseIII and labeled and unlabeled nucleoside triphosphates. This expermimentmonitors the occupancy of class III genes with active transcriptioncomplexes.¹⁹ 5S RNA transcription can easily be assessed since therepetitive 5S genes give rise to a prominent band on a denaturingpolyacrylamide gel. An autoradiogram was taken of the gel and thefollowing observations made based on the observed autoradiogram.¹⁹Schiissel, M. S., Cell, supra

Concentrations of polyamide 1 as low as 100 nM have a pronounced andselective effect on 5S transcription. At higher polyamide concentration,a general decrease in the transcriptional activity of the nuclei isobserved; however, at each concentration tested, the effects of thepolyamide are much greater on 5S RNA transcrption than on tRNAtranscription. Having established that nearly max inhibition of 5Stranscription is achieve d with 1 μM polyamide 1, we monitored nucleartranscription after various times of cell growth in the presence of thepolyamide. No inhibition is observed for zero time incubation withpolyamide 1 at 1 μM concentration, indicating that disruption oftranscription complexes does not occur during or after the isolation orwork-up of cell nuclei. Statistically equivalent levels of 5Stranscription were observed when the cells were exposed to polyamide 1for 24, 48 or 72 hours.

The observations support the conclusion that polyamide 1 is able toenter cells, transit to the nucleus and disrupt transcription complexeson the chromosomal 5S RNA genes. To rule out the possibility that theobserved inhibition is due to some non-specific toxicity of thepolyamide rather than to direct binding to the 5S RNA gene, the effectsof mismatch polyamides 2 and 3 in the nuclear transcription assay weremonitored. Only a small effect on 5S RNA synthesis relative to tRNAsynthesis is observed with 1 AM of the mismatch polyamides 2 or 3 in theculture medium for 24 hours. This result indicates that the generalinhibition of transcription observed with high concentrations ofpolyamide 1 may be a secondary effect of the inhibition of 5S RNAsynthesis in vivo, rather than the result of non-specific polyamideinteractions. Polyamide 2 affects a small enhancement of 5S RNAtranscription in vitro and in vivo, indicating that polyamides may beable to upregulate transcription in certain cases.

As evidenced by the above results, the subject invention provides novelcompounds, which are oligomers of organic cyclic groups, particularlyazoles, where the compounds fit in the minor groove of dsDNA and providefor hydrogen bonding, polar interactions, and van der Waal'sinteractions resulting in high affinities and high associationconstants.

The subject compositions provide for substantial differentiation betweenthe target sequence and single mismatch sequences. Normally, there willbe at least a two-fold difference between the two sequences, moreusually at least a five-fold difference, and preferentially at least aten-fold difference or greater. In this way, one can insure that thetarget sequence will be primarily affected, with little effect on othersequences. Normally, the target sequence will be at least fivenucleotides, usually at least six nucleotides, more usually at leasteight nucleotides, and not more than about twenty nucleotides. By usingcombinations of compositions, where the combinations bind to differentsequences, which may be proximal to each other, one may further enhancethe inhibition at a particular gene.

The subject compositions are shown to bind with high affinities tospecific dsDNA sequences and with substantially lower affinities tosingle base mismatches. In this way, even in complex compositions ofdsDNA, such as may be encountered in cellular compositions, there issubstantial assurance that the target sequence will be affected andother sequences will be little affected, if at all. Furthermore, thesubject compositions are capable of transport across a cellular membraneand through the cytosol to the nucleus. The subject compositions arecapable of binding to chromosomal dsDNA involved with nucleosomes andinhibit transcription of genes which form complexes with the subjectcompositions. Single oligomers may be employed or combinations ofoligomers to provide for the desired complex formation. By using thesubject compositions in diagnosis, one is not required to melt the DNAto provide for single-stranded DNA. Rather, the subject compositions canaccurately target the dsDNA and avoid the melting and competitionbetween the natural strands and the labeled complementary strand, as isemployed conventionally today. The subject compositions may be used forcleavage of dsDNA at specific sites, so as to isolate target DNA, whichmay then be readily amplified using PCR. By further modifing the subjectcompositions, one may further expand their applications in their use foridentifying sequences, cleaving specific sequences, investigating therole of genes, screening for the presence of sequences in cells, andinhibiting proliferation of cells.

The references described throughout this specification are fullyincorporated by reference.

Having now fully described the invention, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the inventionas set forth herein.

What is claimed is:
 1. A method for forming a specific complex betweentarget dsDNA and an oligomer selected to bind at a minor groove site ofsaid target dsDNA at a K_(a) of at least 10⁹ M⁻¹, said oligomercomprising organic cyclic groups of from 5 to 6 ring atoms, where atleast 60% of the cyclic groups are heterocycles having from 1 to 3 ringatoms that are heteroatoms independently selected from the groupconsisting of nitrogen, oxygen and sulfur, and wherein at least 60% ofthe heterocycles have at least one nitrogen ring atom, wherein saidoligomer is defined as comprising at least 6 of said nitrogen containingheterocycles disposed in a non-branching configuration relative to oneanother, where at least one of said heterocycles is specific for A, G, Cor T, said oligomer comprising an internal molecule forming a hairpinturn, and said heterocycles being linked by one or more groups forforming hydrogen bonds to available nitrogen and oxygen atoms of saiddsDNA, said method comprising: bringing together under complex formingconditions, said oligomer with dsDNA, whereby complexes form betweensaid oligomer at said target dsDNA.
 2. A method for forming a specificcomplex between target dsDNA and a first and a second oligomer at aminor groove site, where said first and second oligomers are selected toprovide binding at a K_(a) of at least 10⁹ M⁻¹, said first and secondoligomers comprising organic cyclic groups of from 5 to 6 ring atoms,where at least 60% of the cyclic groups are heterocycles having from 1to 3 ring atoms that are heteroatoms independently selected from thegroup consisting of nitrogen, oxygen and sulfur, and wherein at least60% of the heterocycles have at least one nitrogen ring atom, whereinsaid first and second oligomers are defined as having at least 6 of saidnitrogen containing heterocycles disposed in a non-branchingconfiguration relative to one another, where at least one of saidheterocycles is specific for A, G, C or T, said heterocycles beinglinked by one or more groups for forming hydrogen bonds to availablenitrogen or oxygen atoms, or nitrogen and oxygen atoms of said dsDNA,said method comprising: bringing together under complex formingconditions, said first and second oligomers with dsDNA, wherebycomplexes form between said first and second oligomers and said targetdsDNA.
 3. A method for forming a specific complex between target dsDNAin the minor groove and from 1 to 2 oligomers, where the 1 to 2oligomers are selected to provide binding to said minor groove site,said 1 to 2 oligomers comprised of nitrogen containing heterocyclesconsisting of N-methyl pyrrole (Py) and N-methyl imidazole (Im), whereinsaid nitrogen containing heterocycles and other members of said 1 to 2oligomers are selected to provide a K_(a) of greater than about 10⁹ M⁻¹,wherein each of said oligomers is comprised of at least 6 said nitrogencontaining heterocycles disposed in a non-branching configurationrelative to one another, where the order of nitrogen containingheterocycles in relation to said target dsDNA is defined as Im/Py injuxtaposition to G/C, Py/Im in juxtaposition to CM, and Py/Py injuxtaposition to A/T and T/A, said nitrogen containing heterocyclesbeing linked by one or more linking groups for forming hydrogen bonds toavailable nitrogen or oxygen, or nitrogen and oxygen atoms of saiddsDNA, said method comprising: bringing together under complex formingconditions, said 1 to 2 oligomers with dsDNA, whereby complexes formbetween said 1 to 2 oligomers and any target dsDNA.
 4. A methodaccording to claim 3, wherein said second oligomers have one β-alanineseparating units of at least 2 N-heterocycles.
 5. A method according toclaim 3, wherein at least one of said 1 to 2 oligomers have oneβ-alanine separating units of at least 2 nitrogen containingheterocycles.
 6. A method according to claim 4, wherein there are notmore than 2 consecutive Im nitrogen containing heterocycles.
 7. A methodaccording to claim 4, wherein at least one of said 1 to 2 oligomerscomprise at least 8 nitrogen containing heterocycles.
 8. A methodaccording to claim 3, wherein at least one of said 1 to 2 oligomerscomprises at least 2 unpaired nitrogen containing heterocycles.
 9. Amethod according to claim 7, wherein each of said 1 to 2 oligomerscomprises 3 unpaired nitrogen containing heterocycles.
 10. A methodaccording to claim 3, wherein at least one of said 1 to 2 oligomerscomprises at least 8 nitrogen containing heterocycles.
 11. A methodaccording to claim 8, wherein at least one of said 1 to 2 oligomerscomprises at least 2 unpaired nitrogen containing heterocycles.
 12. Amethod for forming a specific complex between target dsDNA in the minorgroove and a pair of oligomers of N-heterocycles consisting of N-methylpyrrole (Py) and N-methyl imidazole (Im), wherein said N-heterocyclesare selected to provide binding in said minor groove with a K_(a) of atleast 10⁹ M⁻¹, wherein said oligomer is comprised of at least 6 saidheterocycles, where the order of heterocycles in relation to said targetdsDNA is defined as Im/Py in juxtaposition to G/C, Py/Im injuxtaposition to C/G, and Py/Py in juxtaposition to A/T and T/A and acomplementary pair of heterocycles refers to the complementary pair ofnucleotides, said oligomers comprise an internal β-alanine, saidinternal β-alanine being in juxtaposition to A and T and forming acomplementary pair with itself, each oligomer terminating in a glycineor β-alanine amino acid joined to an alkyl chain of from 2 to 4 carbonatoms comprising a polar group, said heterocycles being linked by chainsof oligomers 2 atoms comprising NH groups for forming hydrogen bonds toavailable nitrogen and oxygen atoms of said dsDNA, with the proviso thathydrogen atoms away from the surface of said minor groove may besubstituted with substituents of a total of not greater than 30 carbonatoms, said method comprising: bringing together under complex formingconditions, said oligomers with dsDNA, whereby complexes form betweensaid oligomers and any target dsDNA.
 13. A method for forming a specificcomplex between target dsDNA in the minor groove and an oligomer ofnitrogen containing heterocycles selected from the group consisting ofN-methyl pyrrole (Py) and N-methyl imidazole (Im), wherein said nitrogencontaining heterocycles are selected to provide binding in said minorgroove with a K_(a) of at least 10⁹ M⁻¹, wherein said oligomer iscomprised of at least 6 said nitrogen containing heterocycles disposedin a non-branching configuration relative to one another, where theorder of nitrogen containing heterocycles in relation to said targetdsDNA is defined as Im/Py in juxtaposition to G/C, Py/Im injuxtaposition to C/G, and Py/Py in juxtaposition to A/T and T/A, saidnitrogen containing heterocycles being linked by one or more groups forforming hydrogen bonds to available nitrogen or oxygen atoms, ornitrogen and oxygen atoms of said dsDNA, with the proviso that hydrogenatoms away from the surface of said minor groove may be substituted withsubstituents of a total of not greater than 30 carbon atoms, said methodcomprising: bringing together under complex forming conditions, saidoligomer with dsDNA, whereby complexes form between said oligomer andany target dsDNA.
 14. A method according to claim 13, wherein saidoligomer comprises a polar group, wherein said polar group is a tertiaryamine, with the proviso that only one terminus of an oligomer comprisessaid tertiary amine.
 15. A method according to claim 13, wherein saidoligomer comprises a polar group, wherein said polar group is anhydroxyl group.
 16. A method for forming a specific complex betweentarget dsDNA in the minor groove and a pair of oligomers of nitrogencontaining heterocycles selected from the group consisting of N-methylpyrrole (Py) and N-methyl imidazole (Im), wherein said nitrogencontaining heterocycles are selected to provide binding in said minorgroove with a K_(a) of at least 10⁹ M⁻¹, wherein said oligomer iscomprised of at least 6 said nitrogen containing heterocycles disposedin a non-branching configuration relative to one another, where theorder of nitrogen containing heterocycles in relation to said targetdSDNA is defined as Im/Py in juxtaposition to G/C, Py/Im injuxtaposition to C/G, and Py/Py in juxtaposition to A/T and T/A, saidnitrogen containing heterocycles being linked by one or more groups forforming hydrogen bonds to available nitrogen or oxygen atoms, ornitrogen and oxygen atoms of said dsDNA, with the proviso that hydrogenatoms away from the surface of said minor groove may be substituted withsubstituents of a total of not greater than 30 carbon atoms, said methodcomprising: bringing together under complex forming conditions, saidoligomers with dsDNA, whereby complexes form between said oligomers andany target dsDNA.
 17. A method according to claim 16, wherein saidoligomer comprises at least two β-alanines.
 18. A method for detectingthe presence of target dsDNA in a sample, employing a compositioncomprising an oligomer selected to provide binding to a minor groovesite of said target dsDNA at a K_(a) of at least 10⁹ M⁻¹, said oligomercomprising organic cyclic groups of from 5 to 6 ring atoms, where atleast 60% of the cyclic groups are heterocycles having from 1 to 3 ringatoms that are heteroatoms selected from the group consisting ofnitrogen, oxygen and sulfur, and wherein at least 60% of theheterocycles have at least one nitrogen ring atom, wherein said oligomeris defined as having at least 6 of said nitrogen containing heterocyclesdisposed in a non-branching configuration relative to one another, whereat least one of said heterocycles is specific for A, G, C or T, saidoligomer comprising an internal molecule forming a hairpin turn, saidheterocycles being linked by one or more groups for forming hydrogenbonds to available nitrogen and oxygen atoms of said dsDNA, with theproviso that hydrogen atoms away from the surface of said minor groovemay be substituted with substituents of a total of not greater than 100carbon atoms, and a moiety for detecting said complex, said methodcomprising: combining said composition and said sample under complexforming conditions; and detecting the presence of said target dsDNA insaid sample as a complex with said oligomer by means of said moiety. 19.A method of detecting target dsDNA in a sample employing a compositioncomprising a first and a second oligomer, where the first and secondoligomer are selected to provide binding to a minor groove site of saidtarget dsDNA, said first and second oligomers comprised of nitrogencontaining heterocycles selected from the group consisting of N-methylpyrrole (Py) and N-methyl imidazole (Im), wherein said nitrogencontaining heterocycles and other members of said oligomers are selectedto provide a K_(a) of greater than about 10⁹ M⁻¹, wherein each saidoligomer is comprised of at least 6 said nitrogen containingheterocycles disposed in a non-branching configuration relative to oneanother, where the order of nitrogen containing heterocycles in relationto said target dsDNA is defined as Im/Py in juxtaposition to G/C, Py/Imin juxtaposition to C/G, and Py/Py in juxtaposition to A/T and T/A, saidnitrogen containing heterocycles being linked by one or more groups forforming hydrogen bonds to available nitrogen or oxygen atoms, ornitrogen and oxygen atoms of said dsDNA, with the proviso that hydrogenatoms away from the surface of said minor groove may be substituted withsubstituents of a total of not greater than 100 carbon atoms, at leastone oligomer joined to a moiety for detection of complex formationbetween said target dsDNA and said oligomers, said method comprising:combining said composition and said sample under complex formingconditions; and detecting the presence of said target dsDNA in saidsample as a complex with said oligomers by means of said moiety.
 20. Amethod for isolating target dsDNA from a mixture of dsDNA employing acomposition comprising an oligomer selected to provide binding to saidtarget dsDNA at a K_(d)≦1 nM, said oligomer including organic cyclicgroups of from 5 to 6 ring atoms, where at least 60% of the rings areheterocycles having from 1 to 3 ring atoms that are heteroatoms selectedfrom the group consisting of nitrogen, oxygen and sulfur, and wherein atleast 60% of the heterocycles have at least one nitrogen ring atom,wherein said oligomer is defined as having at least 6 of said nitrogencontaining heterocycles, where at least one of said heterocycles isspecific for A, G, C or T, and a moiety for separating said complex,said heterocycles being linked by one or more groups for forminghydrogen bonds to available nitrogen or oxygen atoms, or nitrogen andoxygen atoms of said dsDNA, said method comprising: combining saidcomposition with said mixture of dsDNA under complex forming conditions;and separating complexes which form by means of said moiety.
 21. Amethod for isolating target dsDNA from a mixture of dsDNA employing acomposition comprising a first oligomer and a second oligomer, wheresaid oligomers are selected to provide binding to said target dsDNA at aK_(a) of at least 10⁹ M⁻¹, said oligomers comprising organic cyclicgroups of from 5 to 6 ring atoms, where at least 60% of the rings areheterocycles having from 1 to 3 ring atoms that are heteroatoms selectedfrom the group consisting of nitrogen, oxygen and sulfur, and wherein atleast 60% of the heterocycles have at least one nitrogen ring atom,wherein each said oligomer is defined as having at least 6 of saidnitrogen containing heterocycles, where at least one of saidheterocycles is specific for A, G, C or T, said heterocycles beinglinked by one or more groups for forming hydrogen bonds to availablenitrogen or oxygen atoms, or nitrogen and oxygen atoms of said dsDNA,and a moiety for detecting said complex, at least one oligomer joined toa moiety for separation of complexes between said target dsDNA and saidoligomers, said method comprising: combining said composition and saidsample under complex forming conditions; and separating complexes whichform by means of said moiety.
 22. A composition comprising: oligomerscomprising nitrogen containing heterocycles selected to provide bindingto a minor groove site of said target dsDNA, wherein said nitrogencontaining heterocycles are selected from a group consisting of N-methylpyrrole (Py) and N-methyl imidazole (Im), wherein said nitrogencontaining heterocycles and other members of said oligomers are selectedto provide a K_(a) of greater than about 10⁹ M⁻¹, wherein said oligomeris comprised of at least 6 said nitrogen containing heterocycles, wherethe order of nitrogen containing heterocycles in relation to said targetdsDNA is defined as Im/Py in juxtaposition to G/C, Py/In injuxtaposition to C/G, and Py/Py in juxtaposition to A/T and T/A, saidoligomer comprising at least two units of consecutive nitrogencontaining heterocycles, said oligomer chosen from the group consistingof an oligomer that forms said complementary pairs with itself and anoligomer that forms complementary pairs with another oligomer, wheresaid oligomer that forms said complementary pairs with itself comprisesan internal γ-aminobutyric acid, and said oligomer that formscomplementary pairs with another oligomer comprises an internalβ-alanine, said internal β-alanine being in juxtaposition to A/T and T/Aand forming a complementary pair with itself, said nitrogen containingheterocycles being linked by one or more groups for forming hydrogenbonds to available nitrogen or oxygen atoms, or nitrogen and oxygenatoms of said dsDNA.
 23. A method for isolating target dsDNA from anextended dsDNA comprising said target dsDNA, said method comprising:bringing together said larger fragment of dsDNA and a pair of oligomersof nitrogen containing heterocycles selected from the group consistingof N-methyl pyrrole (Py) and N-methyl imidazole (Im), wherein saidnitrogen containing heterocycles are selected to provide binding in saidminor groove with a K_(a) of at least 10⁹ M⁻¹, wherein said oligomer iscomprised of at least 6 said nitrogen containing heterocycles, where theorder of nitrogen containing heterocycles in relation to said targetdsDNA is defined as Im/Py in juxtaposition to G/C, Py/Im injuxtaposition to C/G, and Py/Py in juxtaposition to A/T and T/A, saidoligomers comprise an internal β-alanine, said internal β-alanine beingin juxtaposition to A and T and forming a complementary pair withitself, said nitrogen containing heterocycles being linked by one ormore groups for forming hydrogen bonds to available nitrogen or oxygenatoms, or nitrogen and oxygen atoms of said dsDNA, with the proviso thathydrogen atoms away from the surface of said minor groove may besubstituted with substituents of a total of not greater than 30 carbonatoms, and at least one terminal end of an oligomer is a functionalitycapable of cleaving dsDNA, whereby a complex is formed between saidoligomers and said extended dsDNA; and cleaving said extended dsDNA bymeans of said functionality.
 24. A minor-groove binding polyamidereagent which binds to duplex DNA in a sequence specific manner, saidpolyamide reagent comprising an oligomer of nitrogen containingheterocycles selected from the group consisting of N-methyl pyrrole (Py)and N-methyl imidazole (Im), wherein said nitrogen containingheterocycles are selected to provide binding in said minor groove with aK_(a) of at least 10⁹ M⁻¹, wherein said oligomer is composed of at least6 said nitrogen containing heterocycles, where the order of nitrogencontaining heterocycles in relation to said target dsDNA is defined asIm/Py in juxtaposition to G/C, Py/Im in juxtaposition to C/G, and Py/Pyin juxtaposition to A/T and T/A, said oligomer comprising at least twounits of 2 consecutive nitrogen containing heterocycles formingcomplementary pairs with itself, said oligomer comprises an internalγ-aminobutyric acid between said two units, and a unit of 6 nitrogencontaining heterocycles comprises an internal β-alanine, said internalβ-alanine being in juxtaposition to A and T and forming a complementarypair with itself, and has from about one to three chemical moietiesappended thereto, wherein, the chemical moieties confer upon thepolyamide reagent properties which render the polyamide reagent usefulfor a purpose selected from the group consisting of purifying duplex DNAin a sequence specific manner, and detecting duplex DNA in a sequencespecific manner.
 25. A minor groove binding polyamide reagent whichbinds to duplex DNA in specific manner, said polyamide reagentcomprising a pair of oligomers of nitrogen containing heterocyclesselected from the group consisting of N-methyl pyrrole (Py) and N-methylimidazole (Im), wherein said nitrogen containing heterocycles areselected to provide binding in said minor groove with a K_(a) of atleast 10⁹ M⁻¹, wherein each said oligomer is comprised of at least 6said nitrogen containing heterocycles, where the order of nitrogencontaining heterocycles in relation to said target dsDNA is defined asIm/Py in juxtaposition to G/C, Py/Im in juxtaposition to C/G, and Py/Pyin juxtaposition to A/T and T/A, said oligomers comprise an internalβ-alanine, said internal β-alanine being in juxtaposition to A and T andforming a complementary pair with itself, said nitrogen containingheterocycles being linked by one or more groups for forming hydrogenbonds to available nitrogen or oxygen atoms, or nitrogen and oxygenatoms of said dsDNA, and has from about one to three chemical moietiesappended thereto, wherein, the chemical moieties confer upon thepolyamide reagent properties which render the polyamide reagent usefulfor a purpose selected from the group consisting of purifying duplex DNAin a sequence specific manner, and detecting duplex DNA in a sequencespecific manner.
 26. A method of detecting target dsDNA in a sampleemploying a composition comprising a first and a second oligomer, wherefirst and second the oligomers are selected to provide binding to aminor groove site of said target dsDNA, said first and second oligomerscomprised of N-heterocycles consisting of N-methyl pyrrole (Py) andN-methyl imidazole (Im), wherein said N-heterocycles and other membersof said oligomers are selected to provide a K_(a) of greater than about10⁹ M⁻¹, wherein said each oligomer is comprised of at least 6 saidheterocycles, where the order of heterocycles in relation to said targetdsDNA is defined as Im/Py in juxtaposition to G/C, Py/Im injuxtaposition to C/G, and Py/Py in juxtaposition to A/T and T/A and acomplementary pair of heterocycles refers to the complementary pair ofnucleotides, said first and second oligomer each comprising at least twounits of consecutive heterocycles forming complementary pairs with theother oligomer, said oligomers comprising an internal β-alanine, saidinternal β-alanine being in juxtaposition to A/T and T/A and forming acomplementary pair with itself, each said oligomer terminating in aglycine or β-alanine amino acid joined to an alkyl chain of from 2 to 4carbon atoms comprising a polar group, said heterocycles being linked bychains of 2 atoms comprising NH groups for forming hydrogen bonds toavailable nitrogen and oxygen atoms of said dsDNA, with the proviso thathydrogen atoms away from the surface of said minor groove may besubstituted with substituents of a total of not greater than 100 carbonatoms, at least one oligomer joined to a moiety for detection of complexformation between said target dsDNA and said oligomers, said methodcomprising: combining said composition and said sample under complexforming conditions; and detecting the presence of said target dsDNA insaid sample as a complex with said oligomers by means of said moiety.27. A method according to claim 26, wherein said moiety is an enzyme, afluorescer, a chemiluminescer, a solid surface, a hapten which binds toa receptor, or a radioactive isotope.
 28. A method according to claim26, wherein said method further comprises: separating any complex fromany other dsDNA in said sample before detecting said complex.
 29. Amethod according to claim 19, wherein said moiety is an enzyme, afluorescer, a chemiluminescer, a solid surface, a hapten which binds toa receptor, or a radioactive isotope.
 30. A method according to claim19, wherein said method further comprises: separating any complex fromany other dsDNA in said sample before detecting said complex.
 31. Amethod according to claim 30, wherein one of said oligomers or saiddsDNA is bound to a solid surface.
 32. A method according to claim 19,wherein said moietyis biotin or digoxin.
 33. A method according to claim19, wherein said dsDNA is a chromosomal fragment.
 34. A method accordingto claim 21, wherein said moiety is a hapten and said separatingcomprises combining said oligomers and mixture with a receptor for saidhapten bound to a solid surface.
 35. A method according to claim 34,wherein said solid surface comprises particles or a vessel wall.
 36. Acomposition according to claim 22, wherein at least two of said nitrogencontaining heterocycles are linked to one another by a carbamyl linkinggroup.
 37. A composition according to claim 22, wherein said compositioncomprises two oligomers.
 38. A composition according to claim 37,wherein at least one oligomer comprises at least 7 nitrogen containingheterocycles.
 39. A composition according to claim 37, wherein at leastone oligomer comprises a β-alanine internal to six nitrogen containingheterocycles disposed in a non-branching configuration relative to oneanother and wherein said β-alanine is separated from said γ-aminobutyricacid by at least 2 nitrogen containing heterocycles.
 40. A compositionaccording to claim 22, wherein said composition comprises 2 oligomers,each oligomer having a sequence of at least 6 nitrogen containingheterocycles disposed in a non-branching configuration relative to oneanother and comprising a β-alanine internal to said at least 6 nitrogencontaining heterocycles disposed in a non-branching configurationrelative to one another.
 41. A composition according to claim 22,wherein at least one of said oligomers is substituted with a chelatedmetal group.
 42. The method of purifying duplex DNA in a sequencespecific manner, by reversible immobilization, which employs thepolyamide reagent of claim
 41. 43. A minor-groove binding polyamidereagent which binds to duplex DNA in specific manner, said polyamidreagent comprising an oligomer of N-heterocycles selected from the groupconsisting of N-methyl pyrrole (Py) and N-methyl imidazole (Im), whereinsaid N-heterocycles are selected to provide binding in said minor groovewith a K_(a) of at least 10⁹ M⁻¹, wherein said oligomer is composed ofat least 6 said heterocycles, where the order of heterocycles inrelation to said target dsDNA is defined as Im/Py in juxtaposition toG/C, Py/Im in juxtaposition to C/G, and Py/Py in juxtaposition to A/Tand T/A said oligomer comprising at least two units of 2 consecutiveheterocycles forming complementary pairs with itself, said oligomercomprises an internal γ-aminobutyric acid between said two units, and aunit of 6 N-heterocycles comprises an internal β-alanine, said internalβ-alanine being in juxtaposition to A and T and forming a complementarypair with itself, and has from about one to three chemical moietiesappended thereto, wherein, the chemical moieties confer upon thepolyamide reagent properties which render the polyamide reagent usefulfor a purpose selected from the group consisting of purifying duplex DNAin a sequence specific manner, and detecting duplex DNA in a sequencespecific manner.
 44. The method of claim 23, wherein, the chemicalmoieties appended to the polyamide reagent include a portion selectedfrom a group consisting of arylbornic acids, biotins, polyhistidinescomprised of from about 2 to 8 amino acids, haptens to which an antibodybinds, and solid phase supports.
 45. The method of claim 23, wherein,the chemical moieties appended to the polyamide reagent include aportion selected from a group consisting of chromophores, fluorophores,metal ion chelators, enzymes, and other moieties detectable by visual,spectroscopic or electronic means.
 46. The method of claim 45, wherein,the chemical moieties appended to the polyamide reagent include aportion selected from a group consisting of arylboronic acids, biotins,polyhistidines comprised of from about 2 to 8 amino acids, haptens towhich an antibody binds, and solid phase supports.
 47. A minor-groovebinding polyamide reagent which binds to duplex DNA in specific manner,said polyamide reagent comprising a pair of oligomers of N-heterocycleselected from the group consisting of N-methyl pyrrole (Py) and N-methylimidazole (Im), wherein said N-heterocycles are selected to providebinding in said minor groove with a K_(a) of at least 10⁹ M⁻¹, whereineach said oligomer is comprised of at least 6 said heterocycles, wherethe order of heterocycles in relation to said target dsDNA is defined asIm/Py in juxtaposition to G/C, Py/Im in juxtaposition to CIG, and Py/Pyin juxtaposition to A/T and T/A said oligomers comprise an internalβ-alanine, said internal β-alanine being in juxtaposition to A and T andforming a complementary pair with itself, said heterocycles being linkedby one or more groups for forming hydrogen bonds to available nitrogenor oxygen atoms, or nitrogen and oxygen atoms of aid dsDNA, and has fromabout one to three chemical moieties appended thereto, wherein, thechemical moieties confer upon the polyamide reagent properties whichrender the polyamide reagent useful for a purpose selected from thegroup consisting of purifying duplex DNA in a sequence specific manner,and detecting duplex DNA in a sequence specific manner.
 48. The methodof claim 45, wherein, the chemical moieties appended to the polyamidereagent include a portion selected from a group consisting ofchromophores, fluorophores, metal ion chelators, enzymes, and othermoieties detectable by visual, spectroscopic or electronic means. 49.The method of claim 48, wherein, the chemical moieties appended to thepolyamide reagent include a portion selected from a group consisting ofeither arylboronic acids, biotins, polyhistidines comprised of fromabout 2 to 8 amino acids, haptens to which an antibody binds, and solidphase supports.
 50. A method of purifying duplex DNA in a sequencespecific manner, by reversible immobilization, which employs thepolyamide reagent of claim
 25. 51. The method of claim 50, wherein, thechemical moieties appended to the polyamide reagent include a portionselected from a group consisting of either arylboronic acids, biotins,polyhistidines comprised of from about 2 to 8 amino acids, haptens towhich an antibody binds, and solid phase supports.
 52. The method ofdetecting duplex DNA in a sequence specific manner which employs thepolyamide reagent of claim
 25. 53. The method of claim 52, wherein, thechemical moieties appended to the polyamide reagent include a portionselected from a group consisting of either chromophores, fluorophores,metal ion chelators, enzymes, or other moieties detectable by visual,spectroscopic or electronic means.
 54. The method of claim 53, whereinthe chemical moieties appended to the polyamide reagent comprise twofluorophores which act as an energy donor and energy acceptor pair. 55.The method of any of claims 1, 2, 3, 13, 16, 18, 19, 20, 21, and 23,wherein said oligomer is defined as having at least 7 of said nitrogencontaining heterocycles.
 56. The method of any one of claims 1-3, 13,16, 18, 19, 20, 21, and 23 wherein said oligomer or each of saidoligomers terminate in a glycine or β-alanine amino acid joined to analkyl chain of from 2 to 4 carbon atoms comprising a polar group. 57.The composition of claim 22 wherein said oligomer is defined as havingat least 7 of said nitrogen containing heterocycles.
 58. The reagent ofclaim 24 wherein said oligomer is defined as having at least 7 of saidnitrogen containing heterocycles.
 59. The method of claim 48 whereinsaid oligomer is defined as having at least 7 of said nitrogencontaining heterocycles.
 60. The composition of claim 22 wherein saidoligomer or each of said oligomer treminate in a glycine or B-alanineamino acid joined to an alkyl chain of from 2 to 4 carbon atomscomprising a polar group.
 61. The reagent of claim 22 or 25 wherein saidoligomer or each of said oligomer terminate in a glycine or B-alanineamino acid joined to an alkyl chain of from 2 to 4 carbon atomscomprising a polar group.